Modulation of C-reactive protein expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of C-reactive protein. The compositions comprise oligonucleotides, targeted to nucleic acid encoding C-reactive protein. Methods of using these compounds for modulation of C-reactive protein expression and for diagnosis and treatment of disease associated with expression of C-reactive protein are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/969,208, filed Jan. 3, 2008, which is a continuation of U.S.application Ser. No. 10/858,500, filed Jun. 1, 2004 issued as U.S. Pat.No. 7,425,545, Sep. 16, 2008, which claims the benefit of the priorityof U.S. Provisional Patent Application No. 60/475,272, filed Jun. 2,2003, and No. 60/540,042, filed Jan. 28, 2004, each of which isincorporated herein in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0014USC2SEQ.txt, created on Oct. 11, 2010 which is 127 Kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of C-reactive protein.

C-reactive protein (also known as CRP and PTX1) is an essential humanacute-phase reactant produced in the liver in response to a variety ofinflammatory cytokines. The protein, first identified in 1930, is highlyconserved and considered to be an early indicator of infectious orinflammatory conditions. Plasma C-reactive protein levels increase1,000-fold in response to infection, ischemia, trauma, burns, andinflammatory conditions. Since the biological half-life of C-reactiveprotein is not influenced by age, liver or kidney function orpharmacotherapy, it is a reliable biochemical marker for tissuedestruction, necrosis and inflammation and its measurement is widelyused to monitor various inflammatory states, angina pectoris, vascularinsults, end-stage renal disease, rheumatoid arthritis, obesity andatherosclerosis (Arici and Walls, Kidney Int., 2001, 59, 407-414; Gabayand Kushner, N. Engl. J. Med., 1999, 340, 448-454; Highton et al., J.Rheumatol., 1985, 12, 871-875; Hulthe et al., Clin Sci (Colch), 2001,100, 371-378; Lagrand et al., Circulation, 1999, 100, 6+96-102; Morrowand Ridker, Med. Clin. North Am., 2000, 84, 149-161, ix; Szalai et al.,Immunol Res, 1997, 16, 127-136; Westhuyzen and Healy, Ann. Clin. Lab.Sci., 2000, 30, 133-143; Yudkin et al., Atherosclerosis, 2000, 148,209-214).

Improved methods of quantifying C-reactive protein have led to increasedapplication to clinical medicine including diagnoses of urinary tractinfections (Arici and Walls, 2001, cited above), meningitis (Ruuskanenet al., J. Pediatr., 1985, 107, 97-100), neonatal sepsis, erythropoietinresistance (Barany, Nephrol. Dial. Transplant., 2001, 16, 224-227) andoccult bacteremia, conditions in which C-reactive protein is usuallyelevated.

Structurally, C-reactive protein is a member of the pentraxin family ofproteins, which are characterized by a cyclic pentameric structure andradial symmetry. The five identical 24-kDa protomers consist of 206amino acids, and are noncovalently linked (Lei et al., J. Biol. Chem.,1985, 260, 13377-13383; Szalai et al., 1997, cited above). The genomicDNA sequence for human C-reactive protein has been reported by Lei etal. 1985, cited above, as have mutant forms of the protein(International Patent Publication No. WO 96/06624) and methods todeliver materials into cells using the mutant protein as a carrier(International Patent Publication No. WO 00/11207). Polypeptidescorresponding to amino acids 174-185 of C-reactive protein havingimmunomodulatory activity are disclosed and claimed U.S. Pat. No.5,783,179. Peptides corresponding to positions 62-71 of human C-reactiveprotein have also been studied for their ability to inhibit the activityof human leukocyte elastase and/or cathepsin G for the treatment ofinflammatory conditions and these are disclosed in International PatentPublication No. WO 99/00418.

C-reactive protein binds to a broad range of cellular substances such asphosphocholine, fibronectin, chromatin, histones, and ribonucleoproteinin a calcium-dependent manner (Szalai et al., 1997, cited above). It isa ligand for specific receptors on phagocytic leukocytes, mediatesactivation reactions on monocytes and macrophages, and activatescomplement (Szalai et al., 1997, cited above).

The function of C-reactive protein is related to its role in the innateimmune system. Similar to immunoglobulin (Ig) G, it activatescomplement, binds to Fc receptors and acts as an opsonin for variouspathogens. Interaction of C-reactive protein with Fc receptors leads tothe generation of proinflammatory cytokines that enhance theinflammatory response. Unlike IgG, which specifically recognizesdistinct antigenic epitopes, C-reactive protein recognizes altered selfand foreign molecules based on pattern recognition. C-reactive proteinis therefore thought to act as a surveillance molecule for altered selfand certain pathogens. This recognition provides early defense and leadsto a proinflammatory signal and activation of the humoral, adaptiveimmune system. Thus, the C-reactive protein molecule has both arecognition function and an effector function.

The pharmacological modulation of C-reactive protein activity and/or itsexpression is therefore an appropriate point of therapeutic interventionin pathological conditions.

Strategies aimed at modulating C-reactive protein function by targetingprotein levels have involved the use of antibodies, peptides andmolecules that inhibit HMG-CoA reductase.

In a recent trial, it was demonstrated that lovastatin, an inhibitor ofthe enzyme HMG-CoA reductase, is an effective agent in reducing the riskof acute coronary events in participants with elevated C-reactiveprotein levels but no hyperlipidemia; the use of lovastatin resulted ina 14.8 percent reduction in median C-reactive protein levels after oneyear whereas no change was observed in the placebo group (Ridker et al.,N. Engl. J. Med., 2001, 344, 1959-1965). Another statin, cerivastatin,has also been demonstrated to lower C-reactive protein levels inpatients with hypercholesterolemia (Ridker et al., Circulation, 2001,103, 1191-1193.).

However, there are currently no known therapeutic agents thateffectively inhibit C-reactive protein levels and function.Consequently, there remains a long felt need for agents capable ofeffectively and selectively inhibiting C-reactive protein.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for modulatingC-reactive protein expression.

The present invention is directed to compounds, especially nucleic acidand nucleic acid-like oligomers, which are targeted to a nucleic acidencoding C-reactive protein, and which modulate the expression ofC-reactive protein. In particular, this invention relates to compounds,particularly oligonucleotide compounds, which, in preferred embodiments,hybridize with nucleic acid molecules encoding C-reactive protein. Suchcompounds are shown herein to modulate the expression of C-reactiveprotein.

Antisense technology is emerging as an effective means for reducing theexpression of specific gene products and is uniquely useful in a numberof therapeutic, diagnostic, and research applications for the modulationof C-reactive protein expression.

Pharmaceutical and other compositions comprising the compounds of theinvention are also provided. Further provided are methods of screeningfor modulators of C-reactive protein and methods of modulating theexpression of C-reactive protein in cells, tissues or animals comprisingcontacting said cells, tissues or animals with one or more of thecompounds or compositions of the invention. In these methods, the cellsor tissues may be contacted in vivo. Alternatively, the cells or tissuesmay be contacted ex vivo.

Methods of treating an animal, particularly a human, suspected of havingor being prone to a disease or condition associated with expression ofC-reactive protein are also set forth herein. Such methods compriseadministering a therapeutically or prophylactically effective amount ofone or more of the compounds or compositions of the invention to theperson in need of treatment.

In one aspect, the invention provides the use of a compound orcomposition of the invention in the manufacture of a medicament for thetreatment of any and all conditions disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

A. Overview of the Invention

The present invention employs compounds, preferably oligonucleotides andsimilar species for use in modulating the function or effect of nucleicacid molecules encoding C-reactive protein. This is accomplished byproviding oligonucleotides that specifically hybridize with one or morenucleic acid molecules encoding C-reactiVe protein. As used herein, theterms “target nucleic acid” and “nucleic acid molecule encodingC-reactive protein” have been used for convenience to encompass DNAencoding C-reactive protein, RNA (including pre-mRNA and mRNA orportions thereof) transcribed from such DNA, and also cDNA derived fromsuch RNA. The hybridization of a compound of this invention with itstarget nucleic acid is generally referred to as “antisense”.Consequently, the preferred mechanism believed to be included in thepractice of some preferred embodiments of the invention is referred toherein as “antisense inhibition.” Such antisense inhibition is typicallybased upon hydrogen bonding-based hybridization of oligonucleotidestrands or segments such that at least one strand or segment is cleaved,degraded, or otherwise rendered inoperable. In this regard, it ispresently preferred to target specific nucleic acid molecules and theirfunctions for such antisense inhibition.

The functions of DNA to be interfered with can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, and catalyticactivity or complex formation involving the RNA which may be engaged inor facilitated by the RNA. One preferred result of such interferencewith target nucleic acid function is modulation of the expression ofC-reactive protein. In the context of the present invention,“modulation” and “modulation of expression” mean either an increase(stimulation) or a decrease (inhibition) in the amount or levels of anucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition isoften the preferred form of modulation of expression and mRNA is often apreferred target nucleic acid.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,the preferred mechanism of pairing involves hydrogen bonding, which maybe Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases (nucleobases) ofthe strands of oligomeric compounds. For example, adenine and thymineare complementary nucleobases that pair through the formation ofhydrogen bonds. Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleobases of an oligomeric compound. For example,if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases that can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termsthat are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

It is understood in the art that the sequence of an antisense compoundcan be, but need not be, 100% complementary to that of its targetnucleic acid to be specifically hybridizable. Moreover, anoligonucleotide may hybridize over one or more segments such thatintervening or adjacent segments are not involved in the hybridizationevent (e.g., a loop structure or hairpin structure). It is preferredthat the antisense compounds of the present invention comprise at least70%, or at least 75%, or at least 80%, or at least 85% sequencecomplementarity to a target region within the target nucleic acid, morepreferably that they comprise at least 90% sequence complementarity andeven more preferably comprise at least 95% or at least 99% sequencecomplementarity to the target region within the target nucleic acidsequence to which they are targeted. For example, an antisense compoundin which 18 of 20 nucleobases of the antisense compound arecomplementary to a target region, and would therefore specificallyhybridize, would represent 90 percent complementarity. In this example,the remaining noncomplementary nucleobases may be clustered orinterspersed with complementary nucleobases and need not be contiguousto each other or to complementary nucleobases. As such, an antisensecompound which is 18 nucleobases in length having 4 (four)noncomplementary nucleobases which are flanked by two regions ofcomplete complementarity with the target nucleic acid would have 77.8%overall complementarity with the target nucleic acid and would thus fallwithin the scope of the present invention. Percent complementarity of anantisense compound with a region of a target nucleic acid can bedetermined routinely using BLAST programs (basic local alignment searchtools) and PowerBLAST programs known in the art (Altschul et al., J.Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7,649-656).

Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Insome embodiments, homology, sequence identity or complementarity,between the oligomeric and target is between about 50% to about 60%. Insome embodiments, homology, sequence identity or complementarity, isbetween about 60% to about 70%. In some embodiments, homology, sequenceidentity or complementarity, is between about 70% and about 80%. Infurther embodiments, homology, sequence identity or complementarity, isbetween about 80% and about 90%. In further embodiments, homology,sequence identity or complementarity, is about 90%, about 92%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about100%.

B. Compounds of the Invention

According to the present invention, compounds include antisenseoligomeric compounds, antisense oligonucleotides, siRNAs, external guidesequence (EGS) oligonucleotides, alternate splicers and other shortoligomeric compounds that hybridize to at least a portion of the targetnucleic acid. As such, these compounds may be introduced in the form ofsingle-stranded, double-stranded, circular or hairpin oligomericcompounds and may contain structural elements such as internal orterminal bulges or loops. Once introduced to a system, the compounds ofthe invention may elicit the action of one or more enzymes or structuralproteins to effect modification of the target nucleic acid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds that are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While one form of antisense compound is a single-stranded antisenseoligonucleotide, in many species the introduction of double-strandedstructures, such as double-stranded RNA (dsRNA) molecules, inducespotent and specific antisense-mediated reduction of the function of agene or its associated gene products. This phenomenon occurs in bothplants and animals and is believed to have an evolutionary connection toviral defense and transposon silencing.

The first evidence that dsRNA could lead to gene silencing in animalscame in 1995 from work in the nematode, Caenorhabditis elegans (Guo andKempheus, Cell, 1995, 81, 611-620). The primary interference effects ofdsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci.USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanismdefined in Caenorhabditis elegans resulting from exposure todouble-stranded RNA (dsRNA) has since been designated RNA interference(RNAi). This term has been generalized to mean antisense-mediated genesilencing involving the introduction of dsRNA leading to thesequence-specific reduction of endogenous targeted mRNA levels (Fire etal., Nature, 1998, 391, 806-811). Recently, the single-stranded RNAoligomers of antisense polarity of the dsRNAs have been reported to bethe potent inducers of RNAi (Tijsterman et al., Science, 2002, 295,694-697).

In the context of this invention, the term “oligomeric compound” refersto a polymer or oligomer comprising a plurality of monomeric units. Inthe context of this invention, the term “oligonucleotide” refers to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or mimetics, chimeras, analogs and homologs thereof. This termincludes oligonucleotides composed of naturally occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

The oligonucleotides of the present invention also include modifiedoligonucleotides in which a different base is present at one or more ofthe nucleotide positions in the oligonucleotide. For example, if thefirst nucleotide is an adenosine, modified oligonucleotides may beproduced which contain thymidine, guanosine or cytidine at thisposition. This may be done at any of the positions of theoligonucleotide. These oligonucleotides are then tested using themethods described herein to determine their ability to inhibitexpression of C-reactive protein mRNA.

While oligonucleotides are a preferred form of the compounds of thisinvention, the present invention comprehends other families of compoundsas well, including but not limited to oligonucleotide analogs andmimetics such as those described herein.

The compounds in accordance with this invention comprise from about 8 toabout 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides).One of ordinary skill in the art will appreciate that the inventionembodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, or 80 nucleobases in length.

In one embodiment, the compounds of the invention are 12 to 50nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases inlength.

In another embodiment, the compounds of the invention are 15 to 30nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

In another embodiment, the compounds of the invention areoligonucleotides from about 12 to about 50 nucleobases. Furtherembodiments are those comprising from about 15 to about 30 nucleobases.

In another embodiment, the antisense compounds comprise at least 8contiguous nucleobases of an antisense compound disclosed herein.

Antisense compounds 8-80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Exemplary antisense compounds include oligonucleotide sequences thatcomprise at least the 8 consecutive nucleobases from the 5′-terminus ofone of the illustrative preferred antisense compounds (the remainingnucleobases being a consecutive stretch of the same oligonucleotidebeginning immediately upstream of the 5′-terminus of the antisensecompound which is specifically hybridizable to the target nucleic acidand continuing until the oligonucleotide contains about 8 to about 80nucleobases). Similarly preferred antisense compounds are represented byoligonucleotide sequences that comprise at least the 8 consecutivenucleobases from the 3′-terminus of one of the illustrative preferredantisense compounds (the remaining nucleobases being a consecutivestretch of the same oligonucleotide beginning immediately downstream ofthe 3′-terminus of the antisense compound which is specificallyhybridizable to the target nucleic acid and continuing until theoligonucleotide contains about 8 to about 80 nucleobases). Exemplarycompounds of this invention may be found identified in the Examples andlisted in Tables 1, 2 and 3. One having skill in the art armed with thepreferred antisense compounds illustrated herein will be able, withoutundue experimentation, to identify further preferred antisensecompounds.

C. Targets of the Invention

“Targeting” an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes C-reactive protein.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes, having translation initiation codons withthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding C-reactive protein, regardless of thesequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions of amolecule encoding C-reactive protein that may be targeted effectivelywith the antisense compounds of the present invention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region of the moleculeencoding C-reactive protein that may be targeted effectively. Within thecontext of the present invention, a preferred region is the intragenicregion encompassing the translation initiation or termination codon ofthe open reading frame (ORF) of a gene.

Other target regions of molecules encoding C-reactive protein includethe 5′ untranslated region (5′UTR), known in the art to refer to theportion of an mRNA in the 5′ direction from the translation initiationcodon, and thus including nucleotides between the 5′ cap site and thetranslation initiation codon of an mRNA (or corresponding nucleotides onthe gene), and the 3′ untranslated region (3′UTR), known in the art torefer to the portion of an mRNA in the 3′ direction from the translationtermination codon, and thus including nucleotides between thetranslation termination codon and 3′ end of an mRNA (or correspondingnucleotides on the gene). The 5′ cap site of an mRNA comprises anN7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap site. It is also preferred to targetthe 5′ cap region of a molecule encoding C-reactive protein.

Accordingly, the present invention provides antisense compounds thattarget a portion of nucleotides 1-2480 as set forth in SEQ ID NO: 4. Inanother embodiment, the antisense compounds target at least an 8nucleobase portion of nucleotides 1-570, comprising the 5′UTR as setforth in SEQ ID NO: 4. In another embodiment the antisense compoundstarget at least an 8 nucleobase portion of nucleotides 1183-2480comprising the 3′UTR as set forth in SEQ ID NO: 4. In anotherembodiment, the antisense compounds target at least an 8 nucleobaseportion of nucleotides 571-1182 comprising the coding region as setforth in SEQ ID NO: 4. In still other embodiments, the antisensecompounds target at least an 8 nucleobase portion of a “preferred targetsegment” (as defined herein) as set forth in Table 4.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence, resulting in exon-exon junctions at thesites where exons are joined. Targeting exon-exon junctions can beuseful in situations where the overproduction of a normal splice productis implicated in disease, or where the overproduction of an aberrantsplice product is implicated in disease. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also preferred target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sources,known as “fusion transcripts, are also suitable target sites. Intronscan be effectively targeted using antisense compounds targeted to, forexample, DNA or pre-mRNA.

Alternative RNA transcripts can be produced from the same genomic regionof DNA. These alternative transcripts are generally known as “variants”.More specifically, “pre-mRNA variants” are transcripts produced from thesame genomic DNA that differ from other transcripts produced from thesame genomic DNA in either their start or stop position and contain bothintronic and exonic sequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

Variants can be produced through the use of alternative signals to startor stop transcription. Pre-mRNAs and mRNAs can possess more than onestart codon or stop codon. Variants that originate from a pre-mRNA ormRNA that use alternative start codons are known as “alternative startvariants” of that pre-mRNA or mRNA. Those transcripts that use analternative stop codon are known as “alternative stop variants” of thatpre-mRNA or mRNA. One specific type of alternative stop variant is the“polyA variant” in which the multiple transcripts produced result fromthe alternative selection of one of the “polyA stop signals” by thetranscription machinery, thereby producing transcripts that terminate atunique polyA sites. Within the context of the invention, the types ofvariants described herein are also preferred target nucleic acids.

The locations on the target C-reactive protein nucleic acid to which thepreferred antisense compounds hybridize are hereinbelow referred to as“preferred target segments.” As used herein the term “preferred targetsegment” is defined as at least an 8-nucleobase portion of a targetregion of a molecule encoding C-reactive protein to which an activeantisense compound is targeted. While not wishing to be bound by theory,it is presently believed that these target segments represent portionsof the target nucleic acid that are accessible for hybridization.

While the specific sequences of certain preferred C-reactive proteintarget segments are set forth herein, one of skill in the art willrecognize that these serve to illustrate and describe particularembodiments within the scope of the present invention. Additionalpreferred target segments may be identified by one having ordinary skillin view of this specification.

Target segments 8-80 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative preferred target segments of C-reactive protein areconsidered to be suitable for targeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleobases being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 8 to about 80nucleobases). One having skill in the art armed with the preferredtarget segments illustrated herein will be able, without undueexperimentation, to identify further preferred target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

In one embodiment, the oligomeric antisense compounds can be targeted toregions of a target nucleobase sequence, such as those disclosed herein.All regions of a nucleobase sequence to which an oligomeric antisensecompound can be targeted, wherein the regions are greater than or equalto 8 and less than or equal to 80 nucleobases, are described as follows:

Let R(n, n+m−1) be a region from a target nucleobase sequence, where “n”is the 5′-most nucleobase position of the region, where “n+m−1” is the3′-most nucleobase position of the region and where “m” is the length ofthe region. A set “S(m)”, of regions of length “m” is defined as theregions where n ranges from 1 to L−m+1, where L is the length of thetarget nucleobase sequence and L>m. A set, “A”, of all regions can beconstructed as a union of the sets of regions for each length from wherem is greater than or equal to 8 and is less than or equal to 80.

This set of regions can be represented using the following mathematicalnotation:

$A = {{{\bigcup\limits_{m}{{S(m)}\mspace{14mu}{where}\mspace{14mu} m}} \in N}❘{8 \leq m \leq 80}}$

andS(m)={R _(n,n+m−1) |nε{1,2,3, . . . , L−m+1}}

where the mathematical operator 1 indicates “such that”,

where the mathematical operator e indicates “a member of a set” (e.g.yεZ indicates that element y is a member of set Z),

where x is a variable,

where N indicates all natural numbers, defined as positive integers,

and where the mathematical operator ∪ indicates “the union of sets”.

For example, the set of regions for m equal to 8, 9 and 80 can beconstructed in the following manner. The set of regions, each 8nucleobases in length, S(m=8), in a target nucleobase sequence 100nucleobases in length (L=100), beginning at position 1 (n=1) of thetarget nucleobase sequence, can be created using the followingexpression:S(8)={R _(1,8) |nε{1,2,3, . . . , 93}}and describes the set of regions comprising nucleobases 1-8, 2-9, 3-10,4-11, 5-12, 6-13, 7-14, 8-15, 9-16, 10-17, 11-18, 12-19, 13-20, 14-21,15-22, 16-23, 17-24, 18-25, 19-26, 20-27, 21-28, 22-29, 23-30, 24-31,25-32, 26-33, 27-34, 28-35, 29-36, 30-37, 31-38, 32-39, 33-40, 34-41,35-42, 36-43, 37-44, 38-45, 39-46, 40-47, 41-48, 42-49, 43-50, 44-51,45-52, 46-53, 47-54, 48-55, 49-56, 50-57, 51-58, 52-59, 53-60, 54-61,55-62, 56-63, 57-64, 58-65, 59-66, 60-67, 61-68, 62-69, 63-70, 64-71,65-72, 66-73, 67-74, 68-75, 69-76, 70-77, 71-78, 72-79, 73-80, 74-81,75-82, 76-83, 77-84, 78-85, 79-86, 80-87, 81-88, 82-89, 83-90, 84-91,85-92, 86-93, 87-94, 88-95, 89-96, 90-97, 91-98, 92-99, 93-100.

An additional set for regions 20 nucleobases in length, in a targetsequence 100 nucleobases in length, beginning at position 1 of thetarget nucleobase sequence, can be described using the followingexpression:S(20)={R _(1,20) |nε{1,2,3, . . . , 81}}and describes the set of regions comprising nucleobases 1-20, 2-21,3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31, 13-32,14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40, 22-41, 23-42,24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49, 31-50, 32-51, 33-52,34-53, 35-54, 36-55, 37-56, 38-57, 39-58, 40-59, 41-60, 42-61, 43-62,44-63, 45-64, 46-65, 47-66, 48-67, 49-68, 50-69, 51-70, 52-71, 53-72,54-73, 55-74, 56-75, 57-76, 58-77, 59-78, 60-79, 61-80, 62-81, 63-82,64-83, 65-84, 66-85, 67-86, 68-87, 69-88, 70-89, 71-90, 72-91, 73-92,74-93, 75-94, 76-95, 77-96, 78-97, 79-98, 80-99, 81-100.

An additional set for regions 80 nucleobases in length, in a targetsequence 100 nucleobases in length, beginning at position 1 of thetarget nucleobase sequence, can be described using the followingexpression:S(80)={R _(1,80) |nε{1,2,3, . . . , 21}}and describes the set of regions comprising nucleobases 1-80, 2-81,3-82, 4-83, 5-84, 6-85, 7-86, 8-87, 9-88, 10-89, 11-90, 12-91, 13-92,14-93, 15-94, 16-95, 17-96, 18-97, 19-98, 20-99, 21-100.

Thus, in this example, A would include regions 1-8, 2-9, 3-10 . . .93-100, 1-20, 2-21, 3-22 . . . 81-100, 1-80, 2-81, 3-82 . . . 21-100.

The union of these aforementioned example sets and other sets forlengths from 10 to 19 and 21 to 79 can be described using themathematical expression:

$A = {\bigcup\limits_{m}{S(m)}}$

where ∪ represents the union of the sets obtained by combining allmembers of all sets.

The mathematical expressions described herein define all possible targetregions in a target nucleobase sequence of any length L, where theregion is of length m, and where m is greater than or equal to 8 andless than or equal to 80 nucleobases and, and where m is less than L,and where n is less than L−m+1.

In one embodiment, the oligonucleotide compounds of this invention are100% complementary to these sequences or to small sequences found withineach of the above listed sequences. In another embodiment theoligonucleotide compounds have from at least 3 or 5 mismatches per 20consecutive nucleobases in individual nucleobase positions to thesetarget regions. Still other compounds of the invention are targeted tooverlapping regions of the above-identified portions of the C-reactiveprotein sequence.

D. Screening and Target Validation

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of C-reactive protein. “Modulators” are thosecompounds that decrease or increase the expression of a nucleic acidmolecule encoding C-reactive protein and which comprise at least an8-nucleobase portion that is complementary to a preferred targetsegment. The screening method comprises the steps of contacting apreferred target segment of a nucleic acid molecule encoding C-reactiveprotein with one or more candidate modulators, and selecting for one ormore candidate modulators which decrease or increase the expression of anucleic acid molecule encoding C-reactive protein. Once it is shown thatthe candidate modulator or modulators are capable of modulating (e.g.either decreasing or increasing) the expression of a nucleic acidmolecule encoding C-reactive protein, the modulator may then be employedin further investigative studies of the function of C-reactive protein,or for use as a research, diagnostic, or therapeutic agent in accordancewith the present invention.

The preferred target segments of the present invention may be also becombined with their respective complementary antisense compounds of thepresent invention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocesssing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications (Fire et al., Nature,1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons etal., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282,430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95,15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir etal., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15,188-200). For example, such double-stranded moieties have been shown toinhibit the target by the classical hybridization of antisense strand ofthe duplex to the target, thereby triggering enzymatic degradation ofthe target (Tijsterman et al., Science, 2002, 295, 694-697).

The compounds of the present invention can also be applied in the areasof drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between C-reactive protein and a disease state, phenotype, orcondition. These methods include detecting or modulating C-reactiveprotein comprising contacting a sample, tissue, cell, or organism withthe compounds of the present invention, measuring the nucleic acid orprotein level of C-reactive protein and/or a related phenotypic orchemical endpoint at some time after treatment, and optionally comparingthe measured value to a non-treated sample or sample treated with afurther compound of the invention. These methods can also be performedin parallel or in combination with other experiments to determine thefunction of unknown genes for the process of target validation or todetermine the validity of a particular gene product as a target fortreatment or prevention of a particular disease, condition, orphenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention are utilized for diagnostics,therapeutics, prophylaxis and as research reagents and kits.Furthermore, antisense oligonucleotides, which are able to inhibit geneexpression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics, the compounds of the present invention,either alone or in combination with other compounds or therapeutics, areused as tools in differential and/or combinatorial analyses to elucidateexpression patterns of a portion or the entire complement of genesexpressed within cells and tissues.

As used herein the term “biological system” or “system” is defined asany organism, cell, cell culture or tissue that expresses, or is madecompetant to express products of the gene encoding C-reactive protein.These include, but are not limited to, humans, transgenic animals,cells, cell cultures, tissues, xenografts, transplants and combinationsthereof.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundsthat affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The compounds of the invention are useful for research and diagnostics,because these compounds hybridize to nucleic acids encoding C-reactiveprotein. Primers and probes are useful in methods requiring the specificdetection of nucleic acid molecules encoding C-reactive protein and inthe amplification of said nucleic acid molecules for detection or foruse in further studies of C-reactive protein. Hybridization of theprimers and probes disclosed herein with a nucleic acid encodingC-reactive protein can be detected by means known in the art. Such meansmay include conjugation of an enzyme to the primers and probes,radiolabelling of the primers and probes or any other suitable detectionmeans. Kits using such detection means for detecting the level ofC-reactive protein in a sample may also be prepared.

The invention further provides for the use of a compound or compositionof the invention in the manufacture of a medicament for the treatment ofany and all conditions disclosed herein.

Antisense compounds of the invention are provided for the treatment of,or use in the manufacture of a medicament for the treatment of,neurological conditions including obstructive sleep apnea, Alzheimer'sdisease, ALS, Parkinson's disease, various ataxias, and maculardegeneration.

Antisense compounds of the invention are provided for the treatment of,or use in the manufacture of a medicament for the treatment of,metabolic conditions including obesity, metabolic syndrome, anddiabetes.

Antisense compounds of the invention are provided for the treatment of,or use in the manufacture of a medicament for the treatment of,cardiovascular conditions including sudden cardiac death, coronaryartery disease (CAD), unstable angina, stroke, elective stent placement,angioplasty, atherosclerosis, post percutaneous transluminal angioplasty(PTCA), post peripheral vascular disease, post myocardial infarction(MI), cardiac transplantation, hypertension, mitral valvecommissurotomy, thrombosis, deep vein thrombus, end-stage renal disease(ESRD), renal dialysis, complement activation, congestive heart failure,systemic vasculitis, and cardiopulmonary bypass

Antisense compounds of the invention are provided for the treatment of,or use in the manufacture of a medicament for the treatment of, women'shealth conditions including premenstrual syndrome (PMS) anddysmenorhhoea.

Antisense compounds of the invention are provided for the treatment of,or use in the manufacture of a medicament for the treatment of,inflammatory diseases including gingivitis, inflammatory bowel disease,ulcerative colitis, rheumatoid arthritis, osteoarthritis, and axialspondyloarthritis.

Antisense compounds of the invention are provided for the treatment of,or use in the manufacture of a medicament for the treatment of,infectious diseases including HIV-associated rheumatic disorders andbacterial infection.

Antisense compounds of the invention are provided for the treatment of,or use in the manufacture of a medicament for the treatment of,pulmonary conditions including asthma and chronic obstructive pulmonarydisease.

Antisense compounds of the invention are provided for the treatment of,or use in the manufacture of a medicament for the treatment of,musculoskeletal conditions including lower back pain, intense physicalexercise, endurance training, and age-related disorders.

Antisense compounds of the invention are provided for the treatment of,or use in the manufacture of a medicament for the treatment of, cancersincluding pulmonary cancer and colon cancer.

Among diagnostic uses is the measurement of C-reactive protein levels inpatients to identify those who may benefit from a treatment strategyaimed at attenuation of inflammation. Such patients suitable fordiagnosis include patients with coronary artery stenting, e.g., todiagnose tendencies for myocardial infarction or patients with ESRD orother symptoms related to renal disorders, e.g., hypertension, duresis,renal failure.

The specificity and sensitivity of antisense are also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues and animals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder that can be treated by modulating the expression ofC-reactive protein is treated by administering antisense compounds inaccordance with this invention. For example, in one non-limitingembodiment, the methods comprise the step of administering to the animalin need of treatment, a therapeutically effective amount of a C-reactiveprotein inhibitor. The C-reactive protein inhibitors of the presentinvention effectively inhibit the activity of the C-reactive protein orinhibit the expression of the C-reactive protein. For example, such acompound or composition that reduces levels of C-reactive protein isuseful to prevent morbidity and mortality for subjects with acutecoronary syndrome. Such a composition is useful for reducinginflammation mediated by C-reactive protein in a subject, e.g., to treator prevent or reduce the progression of, atherosclerosis; to treat orprevent or reduce the progression of, acute vascular damage atatherosclerotic plaque sites or in coronary arteries; or to treat orprevent or reduce the progression of, damage caused by inflammationassociated with myocardial infarctions or renal inflammation. Stillother therapeutic or prophylactic methods using the C-reactive proteininhibitory compounds of this invention include to treat patients withcoronary artery stenting; or to treat patients with ESRD or other renaldiseases or related inflammatory disorders.

In one embodiment, the activity or expression of C-reactive protein inan animal is inhibited by about 10%. Preferably, the activity orexpression of C-reactive protein in an animal is inhibited by about 30%.More preferably, the activity or expression of C-reactive protein in ananimal is inhibited by 50% or more. Thus, the oligomeric compoundsmodulate expression of C-reactive protein mRNA by at least 10%, by atleast 20%, by at least 25%, by at least 30%, by at least 40%, by atleast 50%, by at least 60%, by at least 70%, by at least 75%, by atleast 80%, by at least 85%, by at least 90%, by at least 95%, by atleast 98%, by at least 99%, or by 100%.

For example, the reduction of the expression of C-reactive protein maybe measured in serum, adipose tissue, liver or any other body fluid,tissue or organ of the animal. Preferably, the cells contained withinsaid fluids, tissues or organs being analyzed contain a nucleic acidmolecule encoding C-reactive protein and/or C-reactive protein itself.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

F. Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

Modified Internucleoside Linkages (Backbones)

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones containing a phosphorusatom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue that may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Modified Sugar and Internucleoside Linkages-Mimetics

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage (i.e. the backbone), of the nucleotide units arereplaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate target nucleic acid. One suchcompound, an oligonucleotide mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Further embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified Sugars

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; —O, S—, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Otherpreferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-O-methoxyethyl (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-methoxyethoxy or 2′-MOE) (Martin etal., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. Afurther preferred modification includes 2′-dimethylaminooxyethoxy, i.e.,a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

Other modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

A further modification of the sugar includes Locked Nucleic Acids (LNAs)in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom ofthe sugar ring, thereby forming a bicyclic sugar moiety. The linkage ispreferably a methylene (—CH₂—)_(n) group bridging the 2′ oxygen atom andthe 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof aredescribed in International Patent Publication Nos. WO 98/39352 and WO99/14226.

Natural and Modified Nucleobases

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.and are presently preferred base substitutions, even more particularlywhen combined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121;5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Conjugates

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992,and U.S. Pat. No. 6,287,860, the entire disclosure of which areincorporated herein by reference. Conjugate moieties include but are notlimited to lipid moieties such as a cholesterol moiety, cholic acid, athioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenyl-butazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999), which is incorporated herein byreference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

Oligomeric compounds used in the compositions of the present inventioncan also be modified to have one or more stabilizing groups that aregenerally attached to one or both termini of oligomeric compounds toenhance properties such as for example nuclease stability. Included instabilizing groups are cap structures. By “cap structure or terminal capmoiety” is meant chemical modifications, which have been incorporated ateither terminus of oligonucleotides (see for example InternationalPatent Publication No. WO 97/26270, incorporated by reference herein).These terminal modifications protect the oligomeric compounds havingterminal nucleic acid molecules from exonuclease degradation, and canhelp in delivery and/or localization within a cell. The cap can bepresent at the 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) orcan be present on both termini. In non-limiting examples, the 5′-capincludes inverted abasic residue (moiety), 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety (for more details seeWincott et al., International PCT publication No. WO 97/26270,incorporated by reference herein).

Particularly preferred 3′-cap structures of the present inventioninclude, for example 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Tyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

Further 3′ and 5′-stabilizing groups that can be used to cap one or bothends of an oligomeric compound to impart nuclease stability includethose disclosed in WO 03/004602 published on Jan. 16, 2003.

Chimeric Compounds

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

The present invention also includes antisense compounds that arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” in thecontext of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Preferred chimeric oligonucleotides are those disclosed in the Examplesherein. Particularly preferred chimeric oligonucleotides are thosereferred to as ISIS 133726, ISIS 133719, ISIS 140177, ISIS 104183, ISIS140180, ISIS 133731, ISIS 140187, ISIS 133712, ISIS 140194, ISIS 133730,and ISIS 133729.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Chimeric antisense compounds can be of several different types. Theseinclude a first type wherein the “gap” segment of linked nucleosides ispositioned between 5′ and 3′ “wing” segments of linked nucleosides and asecond “open end” type wherein the “gap” segment is located at eitherthe 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotidesof the first type are also known in the art as “gapmers” or gappedoligonucleotides. Oligonucleotides of the second type are also known inthe art as “hemimers” or “wingmers”. Such compounds have also beenreferred to in the art as hybrids. In a gapmer that is 20 nucleotides inlength, a gap or wing can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17 or 18 nucleotides in length. In one embodiment, a20-nucleotide gapmer is comprised of a gap 8 nucleotides in length,flanked on both the 5′ and 3′ sides by wings 6 nucleotides in length. Inanother embodiment, a 20-nucleotide gapmer is comprised of a gap 10nucleotides in length, flanked on both the 5′ and 3′ sides by wings 5nucleotides in length. In another embodiment, a 20-nucleotide gapmer iscomprised of a gap 12 nucleotides in length flanked on both the 5′ and3′ sides by wings 4 nucleotides in length. In a further embodiment, a20-nucleotide gapmer is comprised of a gap 14 nucleotides in lengthflanked on both the 5′ and 3′ sides by wings 3 nucleotides in length. Inanother embodiment, a 20-nucleotide gapmer is comprised of a gap 16nucleotides in length flanked on both the 5′ and 3′ sides by wings 2nucleotides in length. In a further embodiment, a 20-nucleotide gapmeris comprised of a gap 18 nucleotides in length flanked on both the 5′and 3′ ends by wings 1 nucleotide in length. Alternatively, the wingsare of different lengths, for example, a 20-nucleotide gapmer may becomprised of a gap 10 nucleotides in length, flanked by a 6-nucleotidewing on one side (5′ or 3′) and a 4-nucleotide wing on the other side(5′ or 3′).

In a hemimer, an “open end” chimeric antisense compound, 20 nucleotidesin length, a gap segment, located at either the 5′ or 3′ terminus of theoligomeric compound, can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18 or 19 nucleotides in length. For example, a20-nucleotide hemimer can have a gap segment of 10 nucleotides at the 5′end and a second segment of 10 nucleotides at the 3′ end. Alternatively,a 20-nucleotide hemimer can have a gap segment of 10 nucleotides at the3′ end and a second segment of 10 nucleotides at the 5′ end.

Representative United States patents that teach the preparation of suchhybrid structures include, but are not limited to, U.S. Pat. Nos.5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

G. Formulations

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, preferred examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration. Pharmaceutical compositionsand formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug that may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes, which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids. When incorporated into liposomes, these specialized lipidsresult in enhanced circulation lifetimes relative to liposomes lackingsuch specialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposomecomprises one or more glycolipids or is derivatized with one or morehydrophilic polymers, such as a polyethylene glycol (PEG) moiety.Liposomes and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Topical formulations are describedin detail in U.S. patent application Ser. No. 09/315,298 filed on May20, 1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein in its entirety. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Oralformulations for oligonucleotides and their preparation are described indetail in U.S. Patent Publication No. 2003/0040497 (Feb. 27, 2003) andits parent applications; U.S. Patent Publication No. 2003/0027780 (Feb.6, 2003) and its parent applications; and U.S. patent application Ser.No. 10/071,822, filed Feb. 8, 2002, each of which is incorporated hereinby reference in their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Oligonucleotides may be formulated for delivery in vivo in an acceptabledosage form, e.g. as parenteral or non-parenteral formulations.Parenteral formulations include intravenous (IV), subcutaneous (SC),intraperitoneal (IP), intravitreal and intramuscular (IM) formulations,as well as formulations for delivery via pulmonary inhalation,intranasal administration, topical administration, etc. Non-parenteralformulations include formulations for delivery via the alimentary canal,e.g. oral administration, rectal administration, intrajejunalinstillation, etc. Rectal administration includes administration as anenema or a suppository. Oral administration includes administration as acapsule, a gel capsule, a pill, an elixir, etc.

In some embodiments, an oligonucleotide may be administered to a subjectvia an oral route of administration. The subject may be an animal or ahuman (man). An animal subject may be a mammal, such as a mouse, a rat,a dog, a guinea pig, a monkey, a non-human primate, a cat or a pig.Non-human primates include monkeys and chimpanzees. A suitable animalsubject may be an experimental animal, such as a mouse, rat, mouse, arat, a dog, a monkey, a non-human primate, a cat or a pig.

In some embodiments, the subject may be a human. In certain embodiments,the subject may be a human patient in need of therapeutic treatment asdiscussed in more detail herein. In certain embodiments, the subject maybe in need of modulation of expression of one or more genes as discussedin more detail herein. In some particular embodiments, the subject maybe in need of inhibition of expression of one or more genes as discussedin more detail herein. In particular embodiments, the subject may be inneed of modulation, i.e. inhibition or enhancement, of C-reactiveprotein in order to obtain therapeutic indications discussed in moredetail herein.

In some embodiments, non-parenteral (e.g. oral) oligonucleotideformulations according to the present invention result in enhancedbioavailability of the oligonucleotide. In this context, the term“bioavailability” refers to a measurement of that portion of anadministered drug, which reaches the circulatory system (e.g. blood,especially blood plasma) when a particular mode of administration isused to deliver the drug. Enhanced bioavailability refers to aparticular mode of administration's ability to deliver oligonucleotideto the peripheral blood plasma of a subject relative to another mode ofadministration. For example, when a non-parenteral mode ofadministration (e.g. an oral mode) is used to introduce the drug into asubject, the bioavailability for that mode of administration may becompared to a different mode of administration, e.g. an IV mode ofadministration. In some embodiments, the area under a compound's bloodplasma concentration curve (AUC₀) after non-parenteral (e.g. oral,rectal, intrajejunal) administration may be divided by the area underthe drug's plasma concentration curve after intravenous (i.v.)administration (AUC_(iv)) to provide a dimensionless quotient (relativebioavailability, RB) that represents fraction of compound absorbed viathe non-parenteral route as compared to the IV route. A composition'sbioavailability is said to be enhanced in comparison to anothercomposition's bioavailability when the first composition's relativebioavailability (RB₁) is greater than the second composition's relativebioavailability (RB₂).

In general, bioavailability correlates with therapeutic efficacy when acompound's therapeutic efficacy is related to the blood concentrationachieved, even if the drug's ultimate site of action is intracellular(van Berge-Henegouwen et al., Gastroenterol., 1977, 73, 300).Bioavailability studies have been used to determine the degree ofintestinal absorption of a drug by measuring the change in peripheralblood levels of the drug after an oral dose (DiSanto, Chapter 76 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 1451-1458).

In general, an oral composition's bioavailability is said to be“enhanced” when its relative bioavailability is greater than thebioavailability of a composition substantially consisting of pureoligonucleotide, i.e. oligonucleotide in the absence of a penetrationenhancer.

Organ bioavailability refers to the concentration of compound in anorgan. Organ bioavailability may be measured in test subjects by anumber of means, such as by whole-body radiography. Organbioavailability may be modified, e.g. enhanced, by one or moremodifications to the oligonucleotide, by use of one or more carriercompounds or excipients, etc. as discussed in more detail herein. Ingeneral, an increase in bioavailability will result in an increase inorgan bioavailability.

Oral oligonucleotide compositions according to the present invention maycomprise one or more “mucosal penetration enhancers,” also known as“absorption enhancers” or simply as “penetration enhancers.”Accordingly, some embodiments of the invention comprise at least oneoligonucleotide in combination with at least one penetration enhancer.In general, a penetration enhancer is a substance that facilitates thetransport of a drug across mucous membrane(s) associated with thedesired mode of administration, e.g. intestinal epithelial membranes.Accordingly it is desirable to select one or more penetration enhancersthat facilitate the uptake of an oligonucleotide, without interferingwith the activity of the oligonucleotide, and in a such a manner theoligonucleotide can be introduced into the body of an animal withoutunacceptable side-effects such as toxicity, irritation or allergicresponse.

Embodiments of the present invention provide compositions comprising oneor more pharmaceutically acceptable penetration enhancers, and methodsof using such compositions, which result in the improved bioavailabilityof oligonucleotides administered via non-parenteral modes ofadministration. Heretofore, certain penetration enhancers have been usedto improve the bioavailability of certain drugs. See Muranishi, Crit.Rev. Ther. Drug Carrier Systems, 1990, 7, 1 and Lee et al., Crit. Rev.Ther. Drug Carrier Systems, 1991, 8, 91. It has been found that theuptake and delivery of oligonucleotides, relatively complex moleculeswhich are known to be difficult to administer to animals and man, can begreatly improved even when administered by non-parenteral means throughthe use of a number of different classes of penetration enhancers.

In some embodiments, compositions for non-parenteral administrationinclude one or more modifications from naturally-occurringoligonucleotides (i.e. full-phosphodiester deoxyribosyl orfull-phosphodiester ribosyl oligonucleotides). Such modifications mayincrease binding affinity, nuclease stability, cell or tissuepermeability, tissue distribution, or other biological orpharmacokinetic property. Modifications may be made to the base, thelinker, or the sugar, in general, as discussed in more detail hereinwith regards to oligonucleotide chemistry. In some embodiments of theinvention, compositions for administration to a subject, and inparticular oral compositions for administration to an animal or humansubject, will comprise modified oligonucleotides having one or moremodifications for enhancing affinity, stability, tissue distribution, orother biological property.

Suitable modified linkers include phosphorothioate linkers. In someembodiments according to the invention, the oligonucleotide has at leastone phosphorothioate linker. Phosphorothioate linkers provide nucleasestability as well as plasma protein binding characteristics to theoligonucleotide. Nuclease stability is useful for increasing the in vivolifetime of oligonucleotides, while plasma protein binding decreases therate of first pass clearance of oligonucleotide via renal excretion. Insome embodiments according to the present invention, the oligonucleotidehas at least two phosphorothioate linkers. In some embodiments, whereinthe oligonucleotide has exactly n nucleosides, the oligonucleotide hasfrom one to n−1 phosphorothioate linkages. In some embodiments, whereinthe oligonucleotide has exactly n nucleosides, the oligonucleotide hasn−1 phosphorothioate linkages. In other embodiments wherein theoligonucleotide has exactly n nucleoside, and n is even, theoligonucleotide has from 1 to n/2 phosphorothioate linkages, or, when nis odd, from 1 to (n−1)/2 phosphorothioate linkages. In someembodiments, the oligonucleotide has alternating phosphodiester (PO) andphosphorothioate (PS) linkages. In other embodiments, theoligonucleotide has at least one stretch of two or more consecutive POlinkages and at least one stretch of two or more PS linkages. In otherembodiments, the oligonucleotide has at least two stretches of POlinkages interrupted by at least on PS linkage.

In some embodiments, at least one of the nucleosides is modified on theribosyl sugar unit by a modification that imparts nuclease stability,binding affinity or some other beneficial biological property to thesugar. In some cases, the sugar modification includes a 2′-modification,e.g. the 2′-OH of the ribosyl sugar is replaced or substituted. Suitablereplacements for 2′-OH include 2′-F and 2′-arabino-F. Suitablesubstitutions for OH include 2′-O-alkyl, e.g. 2-O-methyl, and2′-O-substituted alkyl, e.g. 2′-O-methoxyethyl, 2′-O-aminopropyl, etc.In some embodiments, the oligonucleotide contains at least one2′-modification. In some embodiments, the oligonucleotide contains atleast 2 2′-modifications. In some embodiments, the oligonucleotide hasat least one 2′-modification at each of the termini (i.e. the 3′- and5′-terminal nucleosides each have the same or different2′-modifications). In some embodiments, the oligonucleotide has at leasttwo sequential 2′-modifications at each end of the oligonucleotide. Insome embodiments, oligonucleotides further comprise at least onedeoxynucleoside. In particular embodiments, oligonucleotides comprise astretch of deoxynucleosides such that the stretch is capable ofactivating RNase (e.g. RNase H) cleavage of an RNA to which theoligonucleotide is capable of hybridizing. In some embodiments, astretch of deoxynucleosides capable of activating RNase-mediatedcleavage of RNA comprises about 6 to about 16, e.g. about 8 to about 16consecutive deoxynucleosides. In further embodiments, oligonucleotidesare capable of eliciting cleavage by dsRNAse enzymes.

Oral compositions for administration of non-parenteral oligonucleotidecompositions of the present invention may be formulated in variousdosage forms such as, but not limited to, tablets, capsules, liquidsyrups, soft gels, suppositories, and enemas. The term “alimentarydelivery” encompasses e.g. oral, rectal, endoscopic andsublingual/buccal administration. A common requirement for these modesof administration is absorption over some portion or all of thealimentary tract and a need for efficient mucosal penetration of thenucleic acid(s) so administered.

Delivery of a drug via the oral mucosa, as in the case of buccal andsublingual administration, has several desirable features, including, inmany instances, a more rapid rise in plasma concentration of the drugthan via oral delivery (Harvey, Chapter 35 In: Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990, page 711).

Endoscopy may be used for drug delivery directly to an interior portionof the alimentary tract. For example, endoscopic retrogradecystopancreatography (ERCP) takes advantage of extended gastroscopy andpermits selective access to the biliary tract and the pancreatic duct(Hirahata et al., Gan To Kagaku Ryoho, 1992, 19(10 Suppl.), 1591).Pharmaceutical compositions, including liposomal formulations, can bedelivered directly into portions of the alimentary canal, such as, e.g.,the duodenum (Somogyi et al., Pharm. Res., 1995, 12, 149) or the gastricsubmucosa (Akamo et al., Japanese J. Cancer Res., 1994, 85, 652) viaendoscopic means. Gastric lavage devices (Inoue et al., Artif. Organs,1997, 21, 28) and percutaneous endoscopic feeding devices (Pennington etal., Ailment Pharmacol. Ther., 1995, 9, 471) can also be used for directalimentary delivery of pharmaceutical compositions.

In some embodiments, oligonucleotide formulations may be administeredthrough the anus into the rectum or lower intestine. Rectalsuppositories, retention enemas or rectal catheters can be used for thispurpose and may be preferred when patient compliance might otherwise bedifficult to achieve (e.g., in pediatric and geriatric applications, orwhen the patient is vomiting or unconscious). Rectal administration canresult in more prompt and higher blood levels than the oral route.(Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed.,Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711). Becauseabout 50% of the drug that is absorbed from the rectum will bypass theliver, administration by this route significantly reduces the potentialfor first-pass metabolism (Benet et al., Chapter 1 In: Goodman &Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman etal., eds., McGraw-Hill, New York, N.Y., 1996).

One advantageous method of non-parenteral administration oligonucleotidecompositions is oral delivery. Some embodiments employ variouspenetration enhancers in order to effect transport of oligonucleotidesand other nucleic acids across mucosal and epithelial membranes.Penetration enhancers may be classified as belonging to one of fivebroad categories—surfactants, fatty acids, bile salts, chelating agents,and non-chelating non-surfactants (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92). Accordingly, someembodiments comprise oral oligonucleotide compositions comprising atleast one member of the group consisting of surfactants, fatty acids,bile salts, chelating agents, and non-chelating surfactants. Furtherembodiments comprise oral oligonucleotide comprising at least one fattyacid, e.g. capric or lauric acid, or combinations or salts thereof.Other embodiments comprise methods of enhancing the oral bioavailabilityof an oligonucleotide, the method comprising co-administering theoligonucleotide and at least one penetration enhancer.

Other excipients that may be added to oral oligonucleotide compositionsinclude surfactants (or “surface-active agents”), which are chemicalentities which, when dissolved in an aqueous solution, reduce thesurface tension of the solution or the interfacial tension between theaqueous solution and another liquid, with the result that absorption ofoligonucleotides through the alimentary mucosa and other epithelialmembranes is enhanced. In addition to bile salts and fatty acids,surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92); and perfluorohemical emulsions, such as FC-43 (Takahashi et al., J.Pharm. Phamacol., 1988, 40, 252).

Fatty acids and their derivatives which act as penetration enhancers andmay be used in compositions of the present invention include, forexample, oleic acid, lauric acid, capric acid (n-decanoic acid),myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol),dilaurin, caprylic acid, arachidonicacid, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines and mono-and di-glycerides thereof and/or physiologically acceptable saltsthereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate,linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1; El-Hariri et al., J.Pharm. Pharmacol., 1992, 44, 651).

In some embodiments, oligonucleotide compositions for oral deliverycomprise at least two discrete phases, which phases may compriseparticles, capsules, gel-capsules, microspheres, etc. Each phase maycontain one or more oligonucleotides, penetration enhancers,surfactants, bioadhesives, effervescent agents, or other adjuvant,excipient or diluent. In some embodiments, one phase comprises at leastone oligonucleotide and at lease one penetration enhancer. In someembodiments, a first phase comprises at least one oligonucleotide and atleast one penetration enhancer, while a second phase comprises at leastone penetration enhancer. In some embodiments, a first phase comprisesat least one oligonucleotide and at least one penetration enhancer,while a second phase comprises at least one penetration enhancer andsubstantially no oligonucleotide. In some embodiments, at least onephase is compounded with at least one degradation retardant, such as acoating or a matrix, which delays release of the contents of that phase.In some embodiments, a first phase comprises at least oneoligonucleotide, at least one penetration enhancer, while a second phasecomprises at least one penetration enhancer and a release-retardant. Inparticular embodiments, an oral oligonucleotide comprises a first phasecomprising particles containing an oligonucleotide and a penetrationenhancer, and a second phase comprising particles coated with arelease-retarding agent and containing penetration enhancer.

A variety of bile salts also function as penetration enhancers tofacilitate the uptake and bioavailability of drugs. The physiologicalroles of bile include the facilitation of dispersion and absorption oflipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman &Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman etal., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Variousnatural bile salts, and their synthetic derivatives, act as penetrationenhancers. Thus, the term “bile salt” includes any of the naturallyoccurring components of bile as well as any of their syntheticderivatives. The bile salts of the invention include, for example,cholic acid (or its pharmaceutically acceptable sodium salt, sodiumcholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid(sodium deoxycholate), glucholic acid (sodium glucholate), glycholicacid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(CDCA, sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579).

In some embodiments, penetration enhancers useful in some embodiments ofpresent invention are mixtures of penetration enhancing compounds. Onesuch penetration enhancer is a mixture of UDCA (and/or CDCA) with capricand/or lauric acids or salts thereof e.g. sodium. Such mixtures areuseful for enhancing the delivery of biologically active substancesacross mucosal membranes, in particular intestinal mucosa. Otherpenetration enhancer mixtures comprise about 5-95% of bile acid orsalt(s) UDCA and/or CDCA with 5-95% capric and/or lauric acid.Particular penetration enhancers are mixtures of the sodium salts ofUDCA, capric acid and lauric acid in a ratio of about 1:2:2respectively. Anther such penetration enhancer is a mixture of capricand lauric acid (or salts thereof) in a 0.01:1 to 1:0.01 ratio (molebasis). In particular embodiments capric acid and lauric acid arepresent in molar ratios of e.g. about 0.1:1 to about 1:0.1, inparticular about 0.5:1 to about 1:0.5.

Other excipients include chelating agents, i.e. compounds that removemetallic ions from solution by forming complexes therewith, with theresult that absorption of oligonucelotides through the alimentary andother mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315).Chelating agents of the invention include, but are not limited to,disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1; Buur et al., J. ControlRel., 1990, 14, 43).

As used herein, non-chelating non-surfactant penetration enhancers maybe defined as compounds that demonstrate insignificant activity aschelating agents or as surfactants but that nonetheless enhanceabsorption of oligonucleotides through the alimentary and other mucosalmembranes (Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1). This class of penetration enhancers includes, butis not limited to, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), can be used.

Some oral oligonucleotide compositions also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which may beinert (i.e., does not possess biological activity per se) or may benecessary for transport, recognition or pathway activation or mediation,or is recognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115; Takakura et al., Antisense & Nucl.Acid Drug Dev., 1996, 6, 177).

A “pharmaceutical carrier” or “excipient” may be a pharmaceuticallyacceptable solvent, suspending agent or any other pharmacologicallyinert vehicle for delivering one or more nucleic acids to an animal. Theexcipient may be liquid or solid and is selected, with the plannedmanner of administration in mind, so as to provide for the desired bulk,consistency, etc., when combined with a nucleic acid and the othercomponents of a given pharmaceutical composition. Typical pharmaceuticalcarriers include, but are not limited to, binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose, etc.); fillers (e.g., lactose and other sugars,microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates or calcium hydrogen phosphate, etc.);lubricants (e.g., magnesium stearate, talc, silica, colloidal silicondioxide, stearic acid, metallic stearates, hydrogenated vegetable oils,corn starch, polyethylene glycols, sodium benzoate, sodium acetate,etc.); disintegrants (e.g., starch, sodium starch glycolate, EXPLOTAB™disintegrating agent); and wetting agents (e.g., sodium lauryl sulphate,etc.).

Oral oligonucleotide compositions may additionally contain other adjunctcomponents conventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions maycontain additional, compatible, pharmaceutically-active materials suchas, for example, antipuritics, astringents, local anesthetics oranti-inflammatory agents, or may contain additional materials useful inphysically formulating various dosage forms of the composition ofpresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions of thepresent invention.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents that function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxyco-formycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Alternatively, compositions ofthe invention may contain two or more antisense compounds targeted todifferent regions of the same nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

H. Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 μgto 100 g per kg of body weight, from 0.1 μg to 10 g per kg of bodyweight, from 1.0 μg to 1 g per kg of body weight, from 10.0 μg to 100 mgper kg of body weight, from 100 μg to 10 mg per kg of body weight, orfrom 1 mg to 5 mg per kg of body weight and may be given once or moredaily, weekly, monthly or yearly, or even once every 2 to 20 years.Persons of ordinary skill in the art can easily estimate repetitionrates for dosing based on measured residence times and concentrations ofthe drug in bodily fluids or tissues. Following successful treatment, itmay be desirable to have the patient undergo maintenance therapy toprevent the recurrence of the disease state, wherein the oligonucleotideis administered in maintenance doses, ranging from 0.01 μg to 100 g perkg of body weight, once or more daily, to once every 20 years.

The effects of treatments with therapeutic compositions can be assessedfollowing collection of tissues or fluids from a patient or subjectreceiving said treatments. It is known in the art that a biopsy samplecan be procured from certain tissues without resulting in detrimentaleffects to a patient or subject. In certain embodiments, a tissue andits constituent cells comprise, but are not limited to, blood (e.g.,hematopoietic cells, such as human hematopoietic progenitor cells, humanhematopoietic stem cells, CD34⁺ cells CD4⁺ cells), lymphocytes and otherblood lineage cells, bone marrow, breast, cervix, colon, esophagus,lymph node, muscle, peripheral blood, oral mucosa and skin. In otherembodiments, a fluid and its constituent cells comprise, but are notlimited to, blood, urine, semen, synovial fluid, lymphatic fluid andcerebro-spinal fluid. Tissues or fluids procured from patients can beevaluated for expression levels of the target mRNA or protein.Additionally, the mRNA or protein expression levels of other genes knownor suspected to be associated with the specific disease state, conditionor phenotype can be assessed. mRNA levels can be measured or evaluatedby real-time PCR, Northern blot, in situ hybridization or DNA arrayanalysis. Protein levels can be measured or evaluated by ELISA,immunoblotting, quantitative protein assays, protein activity assays(for example, caspase activity assays) immunohistochemistry orimmunocytochemistry.

Furthermore, the effects of treatment can be assessed by measuringbiomarkers associated with the disease or condition in theaforementioned tissues and fluids, collected from a patient or subjectreceiving treatment, by routine clinical methods known in the art. Thesebiomarkers include but are not limited to: glucose, cholesterol,lipoproteins, triglycerides, free fatty acids and other markers ofglucose and lipid metabolism; liver transaminases, bilirubin, albumin,blood urea nitrogen, creatine and other markers of kidney and liverfunction; interleukins, tumor necrosis factors, intracellular adhesionmolecules, C-reactive protein and other markers of inflammation;testosterone, estrogen and other hormones; tumor markers; vitamins,minerals and electrolytes.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same. Each of the references, GENBAN® accession numbers, andthe like recited in the present application is incorporated herein byreference in its entirety.

EXAMPLES Example 1 Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and InternationalPatent Publication No. WO 02/36743; 5′-O-Dimethoxytrityl-thymidineintermediate for 5-methyl dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N⁴-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O—[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2 Oligonucleotide and Oligonucleoside Synthesis

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides, such as thephosphorothioates and alkylated derivatives.

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 seconds and preceded bythe normal capping step. After cleavage from the CPG column anddeblocking in concentrated ammonium hydroxide at 55° C. (12-16 hours),the oligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inInternational Patent Application Nos. PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, alsoidentified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligo-nucleosides, also identified as amide-4 linked oligonucleosides,as well as mixed backbone compounds having, for instance, alternatingMMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 3 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group, which has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine, which notonly cleaves the oligonucleotide from the solid support but also removesthe acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis. Yet, when subsequentlymodified, this orthoester permits deprotection to be carried out underrelatively mild aqueous conditions compatible with the final RNAoligonucleotide product.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand., 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

RNA antisense compounds (RNA oligonucleotides) of the present inventioncan be synthesized by the methods herein or purchased from DharmaconResearch, Inc (Lafayette, Colo.). Once synthesized, complementary RNAantisense compounds can then be annealed by methods known in the art toform double stranded (duplexed) antisense compounds. For example,duplexes can be formed by combining 30 μl of each of the complementarystrands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90°C., then 1 hour at 37° C. The resulting duplexed antisense compounds canbe used in kits, assays, screens, or other methods to investigate therole of a target nucleic acid.

Example 4 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hours at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphoro-thioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5 Design and Screening of Duplexed Antisense Compounds TargetingC-Reactive Protein

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements can be designed to target C-reactive protein. Thenucleobase sequence of the antisense strand of the duplex comprises atleast an 8-nucleobase portion of an oligonucleotide in Table 1. The endsof the strands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of the dsRNAis then designed and synthesized as the complement of the antisensestrand and may also contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the dsRNAduplex are complementary over the central nucleobases, each havingoverhangs at one or both termini. The antisense and sense strands of theduplex comprise from about 17 to 25 nucleotides, or from about 19 to 23nucleotides. Alternatively, the antisense and sense strands comprise 20,21 or 22 nucleotides.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG (SEQ ID NO: 624) and having a two-nucleobaseoverhang of deoxythymidine (dT) has the following structure (AntisenseSEQ ID NO: 625, Complement SEQ ID NO: 626):

Overhangs can range from 2 to 6 nucleobases and these nucleobases may ormay not be complementary to the target nucleic acid. In anotherembodiment, the duplexes may have an overhang on only one terminus.

In another embodiment, a duplex comprising an antisense strand havingthe same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 624) is prepared withblunt ends (no single stranded overhang) as shown (Antisense SEQ ID NO:624, Complement SEQ ID NO: 627):

The RNA duplex can be unimolecular or bimolecular; i.e., the two strandscan be part of a single molecule or may be separate molecules.

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 μM. Once diluted, 30μL of each strand is combined with 15 μL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 μL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 μM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate C-reactive protein expression.

When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM™-1 reduced-serum medium(Gibco BRL) and then treated with 130 μL of OPTI-MEM™-1 mediumcontaining 12 μg/mL LIPOFECTIN™ reagent (Gibco BRL) and the desiredduplex antisense compound at a final concentration of 200 nM. After 5hours of treatment, the medium is replaced with fresh medium. Cells areharvested 16 hours after treatment, at which time RNA is isolated andtarget reduction measured by RT-PCR.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full-length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis were determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32 +/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis 96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis 96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQapparatus) or, for individually prepared samples, on a commercial CEapparatus (e.g., Beckman P/ACE™ 5000, ABI 270 apparatus). Base andbackbone composition was confirmed by mass analysis of the compoundsutilizing electrospray-mass spectroscopy. All assay test plates werediluted from the master plate using single and multi-channel roboticpipettors. Plates were judged to be acceptable if at least 85% of thecompounds on the plate were at least 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal bovine serum (Invitrogen Corporation, Carlsbad, Calif.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Invitrogen Corporation, Carlsbad, Calif.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#353872) at a density of 7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal bovine serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

Hep3B Cells:

The human hepatoma cell line Hep3B (Hep3B2.1-7) was obtained from theAmerican Type Culture Collection (ATCC Catalog # HB-8064; Manassas,Va.). This cell line was initially derived from a hepatocellularcarcinoma of an 8-yr-old black male. The cells are epithelial inmorphology and are tumorigenic in nude mice. These cells can be inducedto produce C-reactive protein by addition of media containing 1 μMdexamethasone (Sigma-Catalog #D2915 St. Louis, Mo.), 400 U/ml IL1B(Sigma-Catalog #19401) and 200 U/ml IL6 (Sigma-Catalog#I139), accordingto the protocol described by Lozanski, et al., (Cytokine, vol. 8, 1996pp. 534-540). Hep3B cells were routinely cultured in Minimum EssentialMedium (MEM) with Earle's Balanced Salt Solution, 2 mM L-glutamine, 1.5g/L sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodiumpyruvate (ATCC #20-2003, Manassas, Va.) and with 10% heat-inactivatedfetal bovine serum (Invitrogen Corporation, Carlsbad, Calif.). Cellswere routinely passaged by trypsinization and dilution when they reached90% confluence.

In order to determine antisense oligonucleotide inhibition of inducedC-reactive protein, Hep3B cells were plated at a density of 100,000cells into each well of a 6 well plate (Primaria, Franklin N.J.,Catalog#3846) in MEM supplemented with 10% fetal bovine serum andallowed to attach overnight. The next day, cells were induced to produceC-reactive protein for 24 hours in regular media supplemented with afinal concentration of 1 μM dexamethasone, 400 U/ml IL1B and 200 U/mlIL6 as described above. At the end of this induction period, the mediawas removed and cells treated for 4 hrs with 50-150 nM of antisenseoligonucleotide and 3.0-4.5 μg LIPOFECTIN™ reagent in MEM alone (minus)serum supplemented with the three cytokines. At the end of the 4-hourdrug treatment, the media was removed and fresh MEM containing FCS andcytokines was added to each well and allowed to sit for an additional 20hrs. RNA was harvested 24 hrs after treatment with oligonucleotide usingthe QIAGEN® RNeasy (Qiagen Ltd, Valencia, Calif.) procedure andC-reactive protein RNA detected using RT-PCR analysis.

Primary Rat Hepatocytes:

Primary rat hepatocytes were prepared from Sprague-Dawley rats purchasedfrom Charles River Labs (Wilmington, Mass.) and were routinely culturedin DMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (Invitrogen Corporation,Carlsbad, Calif.), 100 units per ml penicillin, and 100 micrograms perml streptomycin (Invitrogen Corporation, Carlsbad, Calif.). Cells werecultured to 80% confluence for use in antisense oligonucleotidetransfection experiments.

Primary Rabbit Hepatocytes:

Primary rabbit hepatocytes from New Zealand White rabbits were purchasedfrom InVitro Technologies (Baltimore, Md.) and were routinely culturedin DMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (Invitrogen Corporation,Carlsbad, Calif.), 100 units per ml penicillin, and 100 micrograms perml streptomycin (Invitrogen Corporation, Carlsbad, Calif.). Primaryrabbit hepatocytes are purchased and transfected at 100% confluency.

Primary Mouse Hepatocytes:

Primary mouse hepatocytes were prepared from Balb/c mice purchased fromCharles River Labs (Wilmington, Mass.) and were routinely cultured inDMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (Invitrogen Corporation,Carlsbad, Calif.), 100 units per ml penicillin, and 100 micrograms perml streptomycin (Invitrogen Corporation, Carlsbad, Calif.). Cells werecultured to 80% confluence for use in antisense oligonucleotidetransfection experiments.

Primary Human Hepatocytes:

Pre-plated primary human hepatocytes were purchased from InVitroTechnologies (Baltimore, Md.). Cells were cultured in high-glucose DMEM(Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetalbovine serum (Invitrogen Corporation, Carlsbad, Calif.), 100 units/mLpenicillin and 100 μg/mL streptomycin (Invitrogen Corporation, Carlsbad,Calif.). Cells were transfected with oligonucleotide upon receipt fromthe vendor.

Primary Cynomolgus Monkey Hepatocytes:

Pre-plated primary Cynomolgus monkey hepatocytes were purchased fromInVitro Technologies (Baltimore, Md.). Cells were cultured inhigh-glucose DMEM (Invitrogen Corporation, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (Invitrogen Corporation,Carlsbad, Calif.), 100 units/mL penicillin and 100 μg/mL streptomycin(Invitrogen Corporation, Carlsbad, Calif.). Cells were treated witholigonucleotide upon receipt from the vendor.

Treatment with Antisense Compounds:

When cells reached 65-75% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium or with serum-freeDMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.) and thentreated with 130 μL of OPTI-MEM™-1 medium containing 3.75 μg/mLLIPOFECTIN™ reagent (Invitrogen Corporation, Carlsbad, Calif.) and thedesired concentration of oligonucleotide. Cells are treated and data areobtained in triplicate. After 4-7 hours of treatment at 37° C., themedium was replaced with fresh medium. Cells were harvested 16-24 hoursafter oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1), which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 10 Analysis of Oligonucleotide Inhibition of C-Reactive ProteinExpression

Antisense modulation of C-reactive protein expression can be assayed ina variety of ways known in the art. For example, C-reactive protein mRNAlevels can be quantitated by, e.g., Northern blot analysis, competitivepolymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-timequantitative PCR is presently preferred. RNA analysis can be performedon total cellular RNA or poly(A)+mRNA. The preferred method of RNAanalysis of the present invention is the use of total cellular RNA asdescribed in other examples herein. Methods of RNA isolation are wellknown in the art. Northern blot analysis is also routine in the art.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7600, 7700, or 7900 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions.

Protein levels of C-reactive protein can be quantitated in a variety ofways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed toC-reactive protein can be identified and obtained from a variety ofsources, such as the MSRS catalog of antibodies (Aerie Corporation,Birmingham, Mich.), or can be prepared via conventional monoclonal orpolyclonal antibody generation methods well known in the art.

Example 11 Design of Phenotypic Assays for the Use of C-Reactive ProteinInhibitors

Once C-reactive protein inhibitors have been identified by the methodsdisclosed herein, the compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of C-reactive protein in health and disease.Representative phenotypic assays, which can be purchased from any one ofseveral commercial vendors, include those for determining cellviability, cytotoxicity, proliferation or cell survival (MolecularProbes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assaysincluding enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences,Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.),cell regulation, signal transduction, inflammation, oxidative processesand apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated withC-reactive protein inhibitors identified from the in vitro studies aswell as control compounds at optimal concentrations which are determinedby the methods described above. At the end of the treatment period,treated and untreated cells are analyzed by one or more methods specificfor the assay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status, which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the genotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the C-reactive proteininhibitors. Hallmark genes, or those genes suspected to be associatedwith a specific disease state, condition, or phenotype, are measured inboth treated and untreated cells.

The cells subjected to the phenotypic assays described herein derivefrom in vitro cultures or from tissues or fluids isolated from livingorganisms, both human and non-human. In certain embodiments, a tissueand its constituent cells comprise, but are not limited to, blood (e.g.,hematopoietic cells, such as human hematopoietic progenitor cells, humanhematopoietic stem cells, CD34⁺ cells CD4⁺ cells), lymphocytes and otherblood lineage cells, bone marrow, brain, stem cells, blood vessel,liver, lung, bone, breast, cartilage, cervix, colon, cornea, embryonic,endometrium, endothelial, epithelial, esophagus, facia, fibroblast,follicular, ganglion cells, glial cells, goblet cells, kidney, lymphnode, muscle, neuron, ovaries, pancreas, peripheral blood, prostate,skin, skin, small intestine, spleen, stomach, testes and fetal tissue.In other embodiments, a fluid and its constituent cells comprise, butare not limited to, blood, urine, synovial fluid, lymphatic fluid andcerebro-spinal fluid. The phenotypic assays may also be performed ontissues treated with C-reactive protein inhibitors ex vivo.

Example 12 RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem.,1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation areroutine in the art. Briefly, for cells grown on 96-well plates, growthmedium was removed from the cells and each well was washed with 200 μLcold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 MNaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added toeach well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 150 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 150 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 1 minute. 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and incubatedfor 15 minutes and the vacuum was again applied for 1 minute. Anadditional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE wasthen added to each well of the RNEASY 96™ plate and the vacuum appliedfor a period of 90 seconds. The Buffer RPE wash was then repeated andthe vacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC™ manifold and blotted dry on paper towels. Theplate was then re-attached to the QIAVAC™ manifold fitted with acollection tube rack containing 1.2 mL collection tubes. RNA was theneluted by pipetting 140 μL of RNAse free water into each well,incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN® BIO-ROBOT™ 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-Time Quantitative PCR Analysis of C-Reactive ProteinmRNA Levels

Quantitation of C-reactive protein mRNA levels was accomplished byreal-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. This is a closed-tube,non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR in which amplification productsare quantitated after the PCR is completed, products in real-timequantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 5′ end of the probe and a quencherdye (e.g., TAMRA, obtained from either PE-Applied Biosystems, FosterCity, Calif., Operon Technologies Inc., Alameda, Calif. or IntegratedDNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ Sequence Detection System. In each assay, aseries of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

Gene target quantities are obtained by reverse-transcriptase, real-timePCR. Prior to the real-time PCR, isolated RNA is subjected to a reversetranscriptase (RT) reaction, for the purpose of generating complementaryDNA (cDNA). Reverse transcriptase and real-time PCR reagents wereobtained from Invitrogen Corporation, (Carlsbad, Calif.). RT, real-timePCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCRbuffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP, dCTP anddGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe,4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reversetranscriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL totalRNA solution (20-200 ng). The RT reaction was carried out by incubationfor 30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension). This method of obtaining gene targetquantities is herein referred to as real-time PCR.

Gene target quantities obtained by real-time PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ reagent(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real-time PCR by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).Methods of RNA quantification by RiboGreen™ reagent are taught in Jones,L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 reader (PE Applied Biosystems) with excitation at485 nm and emission at 530 nm.

Probes and primers to human C-reactive protein were designed tohybridize to a human C-reactive protein sequence, using publishedsequence information (GENBANK® accession number M11725.1, incorporatedherein as SEQ ID NO: 4). For human C-reactive protein the PCR primerswere:

-   forward primer: TGACCAGCCTCTCTCATGCTT (SEQ ID NO: 5)-   reverse primer: TCCGACTCTTTGGGAAACACA (SEQ ID NO: 6) and the PCR    probe was: FAM-TGTCGAGGAAGGCTT-TAMRA (SEQ ID NO: 7) where FAM is the    fluorescent dye and TAMRA is the quencher dye. For human GAPDH the    PCR primers were:-   forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO: 8)-   reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 9) and the PCR    probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10)    where JOE is the fluorescent reporter dye and TAMRA is the quencher    dye.

Probes and primers to rat C-reactive protein were designed to hybridizeto a rat C-reactive protein sequence, using published sequenceinformation (GENBANK® accession number M83176.1, incorporated herein asSEQ ID NO: 11). For rat C-reactive protein the PCR primers were:

-   forward primer: AAGCACCCCCAATGTCACC (SEQ ID NO: 12)-   reverse primer: GGGATGGCAGAGCCAATGTA (SEQ ID NO: 13) and the PCR    probe was: FAM-TCCTGGATTCAAGCTTCTATGTGCCTTCA-TAMRA (SEQ ID NO: 14)    where FAM is the fluorescent reporter dye and TAMRA is the quencher    dye. For rat GAPDH the PCR primers were:-   forward primer: TGTTCTAGAGACAGCCGCATCTT (SEQ ID NO: 15)-   reverse primer: CACCGACCTTCACCATCTTGT (SEQ ID NO: 16) and the PCR    probe was: 5′ JOE-TTGTGCAGTGCCAGCCTCGTCTCA-TAMRA 3′ (SEQ ID NO: 17)    where JOE is the fluorescent reporter dye and TAMRA is the quencher    dye.

Example 14 Northern Blot Analysis of C-Reactive Protein mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ reagent (TEL-TEST “B”Inc., Friendswood, Tex.). Total RNA was prepared followingmanufacturer's recommended protocols. Twenty micrograms of total RNA wasfractionated by electrophoresis through 1.2% agarose gels containing1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon,Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes(Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillarytransfer using a Northern/Southern Transfer buffer system (TEL-TEST “B”Inc., Friendswood, Tex.). RNA transfer was confirmed by UVvisualization. Membranes were fixed by UV cross-linking using aSTRATALINKER™ UV Crosslinker 2400 instrument (Stratagene, Inc, La Jolla,Calif.) and then probed using QUICKHYB™ hybridization solution(Stratagene, La Jolla, Calif.) using manufacturer's recommendations forstringent conditions.

To detect human C-reactive protein, a human C-reactive protein specificprobe was prepared by PCR using the forward primer TGACCAGCCTCTCTCATGCTT(SEQ ID NO: 5) and the reverse primer TCCGACTCTTTGGGAAACACA (SEQ ID NO:6). To normalize for variations in loading and transfer efficiencymembranes were stripped and probed for human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

To detect rat C-reactive protein, a rat C-reactive protein specificprobe was prepared by PCR using the forward primer TGACCAGCCTCTCTCATGCTT(SEQ ID NO: 12) and the reverse primer TCCGACTCTTTGGGAAACACA (SEQ ID NO:13). To normalize for variations in loading and transfer efficiencymembranes were stripped and probed for rat glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ apparatus and IMAGEQUANT™ Software V3.3 (MolecularDynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels inuntreated controls.

Example 15 Antisense Inhibition of Human C-Reactive Protein Expressionby Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap

In accordance with the present invention, a series of antisensecompounds was designed to target different regions of the humanC-reactive protein RNA, using published sequences (GENBANK® accessionnumber M11725.1, incorporated herein as SEQ ID NO: 4). The compounds areshown in Table 1. “Target site” indicates the first (5′-most) nucleotidenumber on the particular target sequence to which the compound binds.All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-O-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human C-reactive protein mRNA levels by quantitativereal-time PCR as described in other examples herein. Data, shown inTable 1, are averages from three experiments in which cytokine-inducedHep3B cells were treated with 150 nM of the antisense oligonucleotidesof the present invention. The positive control for each data point isidentified in the table by sequence ID number. If present, “N.D.”indicates “no data”.

TABLE 1 Inhibition of human C-reactive protein mRNA levelsby chimeric phosphorothioate oligonucleotides having2′-MOE wings and a deoxy gap TARGET SEQ ID TARGET % SEQ ISIS # REGION NOSITE SEQUENCE INHIB ID NO 133709 5′UTR 4 16 gcaggtgtcagagcttcggg 77 19133710 5′UTR 4 71 gcagtaagggagtttgcgcc 71 20 133711 5′UTR 4 181gcctgaattcactcctttgg 87 21 133712 Start Codon 4 221 agcttctccatggtcacgtc92 22 133713 Coding 4 281 tggcccttacctgtctggcc 88 23 133714 Intron 4 311ctcagatcaaaactctccca 30 24 133715 Intron 4 341 ttcatgcagtcttagacccc N.D.25 133716 Coding 4 551 gtctgtgagccagaaaaaca 77 26 133717 Coding 4 701cgagaaaatactgtacccac 82 27 133718 Coding 4 781 gacccacccactgtaaaact 8228 133719 Coding 4 871 cagaactccacgatccctga 96 29 133720 Coding 4 1091attaggactgaagggcccgc 86 30 133721 Stop Codon 4 1171 agctggcctcagggccacag80 31 133722 3′UTR 4 1191 gaggtaccttcaggacccac 89 32 133723 3′UTR 4 1361cccagaccagacactttacc 88 33 133724 3′UTR 4 1391 tggaccatttcccagcatag 6734 133725 3′UTR 4 1631 ttctgagactgaagagccct 27 35 133726 3′UTR 4 1671gcactctggacccaaaccag 96 36 133727 3′UTR 4 1711 caggagacctgggcccagca 8537 133728 3′UTR 4 1918 cccagaagagccataaaatt 27 38 133729 3′UTR 4 1961attcacagccccacaaggtt 90 39 133730 3′UTR 4 2161 agaagatgtctcactcccaa 9140 133731 3′UTR 4 2291 tgtttgtcaatcccttggct 93 41 133732 3′UTR 4 2431ttctaaagcaactatcagaa 64 42 140167 5′UTR 4 111 gccttagagctacctcctcc 70 43140168 5′UTR 4 201 ctgctgccagtgatacaagg 69 44 140169 Intron 4 317ccatacctcagatcaaaact 48 45 140170 Intron 4 451 accccttctccagttacaca 6946 140171 Coding 4 671 cagttccgtgtagaagtgga 43 47 140172 Coding 4 761gtatcctatatccttagacc N.D. 48 140173 Coding 4 821 tggagctactgtgacttcag 8249 140174 Coding 4 861 cgatccctgaggcggactcc N.D. 50 140175 Coding 4 901ctcttcctcaccctgggctt 84 51 140176 Coding 4 921 cagtgtatcccttcttcaga 6852 140177 Coding 4 951 gccccaagatgatgcttgct 95 53 140178 Coding 4 1031gtcccacatgttcacatttc 61 54 140179 Coding 4 1111 agtgcccgccagttcaggac 8655 140180 Coding 4 1141 gtgaacacttcgccttgcac 94 56 140181 3′UTR 4 1341tccattctcaggcgctgagg 85 57 140182 3′UTR 4 1461 gaaattatctccaagatctg 3358 140183 3′UTR 4 1551 cagcgcttccttctcagctc 94 59 140184 3′UTR 4 1611gtgaatgtgggcaatgctcc 58 60 140185 3′UTR 4 1651 acacctggccagtgtcctga N.D.61 140186 3′UTR 4 1771 cctttccagtgtgctttgag N.D. 62 140187 3′UTR 4 1831tagtgcttcattttgctctg 93 63 140188 3′UTR 4 1971 tgaagaaagaattcacagcc 5864 140189 3′UTR 4 2041 ggctcctctgacaggacacc 86 65 140190 3′UTR 4 2101gctaggaacacgtaactatc 71 66 140191 3′UTR 4 2121 ggaagactgtagttggtcct 3567 140192 3′UTR 4 2211 ctactggtggtcccaggttc 77 68 140193 3′UTR 4 2271cctccacttccagtttggct 77 69 140194 3′UTR 4 2341 ctggttccagacaaggctga 9270 140195 3′UTR 4 2402 gactcactcaagtaaacagg 71 71 140196 3′UTR 4 2461ttcaaaggtcatagagaagt 28 72

As shown in Table 1, SEQ ID NOs 19, 20, 21, 22, 23, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 36, 37, 39, 40, 41, 42, 43, 44, 46, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, 64, 65, 66, 68, 69, 70 and71 demonstrated at least 50% inhibition of human C-reactive proteinexpression in this assay and are therefore preferred. More preferred areSEQ ID NOs 36, 22 and 56. The target regions to which these preferredsequences are complementary are herein referred to as “preferred targetsegments” and are therefore preferred for targeting by compounds of thepresent invention. These preferred target segments are shown in Table 4.These sequences are shown to contain thymine (T) but one of skill in theart will appreciate that thymine (T) is generally replaced by uracil (U)in RNA sequences. The sequences represent the reverse complement of thepreferred antisense compounds shown in Table 1. “Target site” indicatesthe first (5′-most) nucleotide number on the particular target nucleicacid to which the oligonucleotide binds. Also shown in Table 4 is thespecies in which each of the preferred target segments was found.

In further embodiment of the present invention, a second series ofantisense compounds was designed to target different regions of thehuman C-reactive protein RNA, using published sequences (GENBANK®accession number M11725.1, incorporated herein as SEQ ID NO: 4). Thecompounds are shown in Table 2. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe compound binds. All compounds in Table 2 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-O-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

The compounds were analyzed for their effect on human C-reactive proteinmRNA levels by quantitative real-time PCR using a second set of probesand primer designed to hybridize to a human C-reactive protein sequence,using published sequence information (GENBANK® accession numberM11725.1, incorporated herein as SEQ ID NO: 4). For human C-reactiveprotein the PCR primers were: forward primer: GCTTCCCCTCTTCCCGAA (SEQ IDNO: 73) reverse primer: TGCGCCACTATGTAAATAATTTTCC (SEQ ID NO: 74) andthe PCR probe was: FAM-TCTGACACCTGCCCCAACAAGCAATG-TAMRA (SEQ ID NO: 75)where FAM is the fluorescent dye and TAMRA is the quencher dye. Data,shown in Table 2, are averages from three experiments in whichcytokine-induced Hep3B cells were treated with 150 nM of the antisenseoligonucleotides of the present invention. The positive control for eachdatapoint is identified in the table by sequence ID number. If present,“N.D.” indicates “no data”.

TABLE 2 Inhibition of human C-reactive protein mRNA levelsby chimeric phosphorothioate oligonucleotides having2′-MOE wings and a deoxy gap TARGET CONTROL SEQ ID TARGET % SEQ IDSEQ ID ISIS # REGION NO SITE SEQUENCE INHIB NO NO 140185 3′ UTR 4 1651acacctggccagtgtcctga 37 61 1 140186 3′ UTR 4 1771 cctttccagtgtgctttgag 162 1 329883 3′ UTR 4 10 gtcagagcttcgggaagagg 6 76 1 329884 3′ UTR 4 37tttccaacattgcttgttgg 0 77 1 329885 3′ UTR 4 47 tgtaaataattttccaacat 4178 1 329886 3′ UTR 4 57 tgcgccactatgtaaataat 7 79 1 329887 3′ UTR 4 67taagggagtttgcgccacta 40 80 1 329888 3′ UTR 4 77 tccaaagcagtaagggagtt 2181 1 329889 3′ UTR 4 87 tggatttatatccaaagcag N.D. 82 329890 3′ UTR 4 94tcctgcctggatttatatcc 8 83 1 329891 3′ UTR 4 107 tagagctacctcctcctgcc 184 1 329892 3′ UTR 4 122 ccagatctcttgccttagag 70 85 1 329893 3′ UTR 4132 gctagaagtcccagatctct 38 86 1 329894 3′ UTR 4 157gatgtattcggctgaaagtt 29 87 1 329895 3′ UTR 4 167 ctttggaaaagatgtattcg 2288 1 329896 3′ UTR 4 191 tgatacaagggcctgaattc 30 89 1 329897 Start codon4 206 acgtcctgctgccagtgata 44 90 1 329898 Coding 4 226acaacagcttctccatggtc 43 91 1 329899 Coding 4 231 gaaacacaacagcttctcca 2892 1 329900 Coding 4 241 tcaagaccaagaaacacaac 0 93 1 329901 Coding 4 251gagaggctggtcaagaccaa 15 94 1 329902 Coding 4 258 agcatgagagaggctggtca 5495 1 329903 Coding 4 268 tctggccaaaagcatgagag 48 96 1 329904 Coding 4278 cccttacctgtctggccaaa 45 97 1 329905 Coding 4 283ggtggcccttacctgtctgg 12 98 1 329906 Coding 4 318 cccatacctcagatcaaaac 099 1 329907 Coding 4 342 gttcatgcagtcttagaccc 21 100 1 329908 Coding 4347 agactgttcatgcagtctta N.D. 101 329909 Coding 4 351tttgagactgttcatgcagt 28 102 1 329910 Coding 4 381 gttctgttcatacagtcttt16 103 1 329911 Coding 4 386 ccactgttctgttcatacag 0 104 1 329912 Coding4 391 atgctccactgttctgttca 4 105 1 329913 Coding 4 396gaaggatgctccactgttct 0 106 1 329914 Coding 4 401 accatgaaggatgctccact 49107 1 329915 Coding 4 406 cacacaccatgaaggatgct 33 108 1 329916 Coding 4411 acacacacacaccatgaagg 0 109 1 329917 Coding 4 449cccttctccagttacacacc 3 110 1 329918 Coding 4 459 acagactgaccccttctcca 19111 1 329919 Coding 4 469 agattgagaaacagactgac 52 112 1 329920 Coding 4479 atagaatttaagattgagaa 8 113 1 329921 Coding 4 489tcacttacgtatagaattta 0 114 1 329922 Coding 4 492 ccctcacttacgtatagaat 40115 1 329923 Coding 4 499 atctatcccctcacttacgt 23 116 1 329924 Coding 4510 agatcacacagatctatccc 6 117 1 329925 Coding 4 520gaggtttctcagatcacaca 0 118 1 329926 Coding 4 530 gcaaatgtgagaggtttctc 4119 1 329927 Coding 4 557 cgacatgtctgtgagccaga 39 120 1 329928 Coding 4562 ttcctcgacatgtctgtgag 52 121 1 329929 Coding 4 567aagccttcctcgacatgtct 81 122 1 329930 Coding 4 596 ggaagtatccgactctttgg39 123 1 329931 Coding 4 605 ggatacataggaagtatccg 0 124 1 329932 Coding4 615 gtgctttgagggatacatag 12 125 1 329933 Coding 4 625ttcgttaacggtgctttgag 0 126 1 329934 Coding 4 635 tttgagaggcttcgttaacg 0127 1 329935 Coding 4 645 cagtgaaggctttgagaggc 1 128 1 329936 Coding 4655 tggaggcacacagtgaaggc 69 129 1 329937 Coding 4 660agaagtggaggcacacagtg 0 130 1 329938 Coding 4 665 cgtgtagaagtggaggcaca 36131 1 329939 Coding 4 675 aggacagttccgtgtagaag 40 132 1 329940 Coding 4685 ccacgggtcgaggacagttc 46 133 1 329941 Coding 4 695aatactgtacccacgggtcg 26 134 1 329942 Coding 4 716 tctcttggtggcatacgaga55 135 1 329943 Coding 4 726 cattgtcttgtctcttggtg 70 136 1 329944 Coding4 736 atgagaatctcattgtcttg 58 137 1 329945 Coding 4 746agaccaaaatatgagaatct 6 138 1 329946 Coding 4 756 ctatatccttagaccaaaat 26139 1 329947 Coding 4 765 aactgtatcctatatcctta 0 140 1 329948 Coding 4775 cccactgtaaaactgtatcc 26 141 1 329949 Coding 4 785ttcagacccacccactgtaa N.D. 142 329950 Coding 4 796 tcgaataatatttcagaccc37 143 1 329951 Coding 4 806 ttcaggaacctcgaataata 14 144 1 329952 Coding4 816 ctactgtgacttcaggaacc 59 145 1 329953 Coding 4 826tgtactggagctactgtgac 39 146 1 329954 Coding 4 836 tgtacaaatgtgtactggag60 147 1 329955 Coding 4 846 actcccagcttgtacaaatg 21 148 1 329956 Coding4 856 cctgaggcggactcccagct 62 149 1 329957 Coding 4 860gatccctgaggcggactccc 66 150 1 329958 Coding 4 870 agaactccacgatccctgag30 151 1 329959 Coding 4 880 ccatctacccagaactccac 22 152 1 329960 Coding4 890 cctgggcttcccatctaccc 34 153 1 329961 Coding 4 900tcttcctcaccctgggcttc 52 154 1 329962 Coding 4 910 ttcttcagactcttcctcac38 155 1 329963 Coding 4 920 agtgtatcccttcttcagac 39 156 1 329964 Coding4 944 gatgatgcttgcttctgccc 55 157 1 329965 Coding 4 964gaatcctgctcctgccccaa 37 158 1 329966 Coding 4 967 aaggaatcctgctcctgccc55 159 1 329967 Coding 4 977 gttcccaccgaaggaatcct 26 160 1 329968 Coding4 987 ttccttcaaagttcccaccg 59 161 1 329969 Coding 4 1000accagggactggcttccttc 71 162 1 329970 Coding 4 1010 aatgtctcccaccagggact7 163 1 329971 Coding 4 1020 tcacatttccaatgtctccc 56 164 1 329972 Coding4 1030 tcccacatgttcacatttcc 49 165 1 329973 Coding 4 1040cagcacaaagtcccacatgt 66 166 1 329974 Coding 4 1050 catctggtgacagcacaaag65 167 1 329975 Coding 4 1060 gtgttaatctcatctggtga 47 168 1 329976Coding 4 1070 aagatagatggtgttaatct 37 169 1 329977 Coding 4 1097caggacattaggactgaagg 53 170 1 329978 Coding 4 1107 cccgccagttcaggacatta52 171 1 329979 Coding 4 1117 tacttcagtgcccgccagtt 49 172 1 329980Coding 4 1127 ttgcacttcatacttcagtg 69 173 1 329981 Coding 4 1137acacttcgccttgcacttca 54 174 1 329982 Coding 4 1147 ggtttggtgaacacttcgcc55 175 1 329983 3′ UTR 4 1193 gggaggtaccttcaggaccc 48 176 1 329984 3′UTR 4 1235 taccagagacagagacgtgg 62 177 1 329985 3′ UTR 4 1245aagcgggaggtaccagagac 62 178 1 329986 3′ UTR 4 1283 gcccagagacagagacgtgg68 179 1 329987 3′ UTR 4 1293 gggaacaaaggcccagagac 59 180 1 329988 3′UTR 4 1326 tgaggagggtggagcaggcc 44 181 1 329989 3′ UTR 4 1338attctcaggcgctgaggagg 44 182 1 329990 3′ UTR 4 1348 ctttacctccattctcaggc74 183 1 329991 3′ UTR 4 1358 agaccagacactttacctcc 29 184 1 329992 3′UTR 4 1368 acgagctcccagaccagaca 70 185 1 329993 3′ UTR 4 1378agcatagttaacgagctccc 64 186 1 329994 3′ UTR 4 1388 accatttcccagcatagtta34 187 1 329995 3′ UTR 4 1398 attcttttggaccatttccc 35 188 1 329996 3′UTR 4 1408 tcaaattctgattcttttgg 27 189 1 329997 3′ UTR 4 1451ccaagatctgtccaacttga 55 190 1 329998 3′ UTR 4 1471 tgtgaggtaagaaattatct21 191 1 329999 3′ UTR 4 1481 ttctcatctatgtgaggtaa 74 192 1 330000 3′UTR 4 1491 ggtgttagttttctcatcta 63 193 1 330001 3′ UTR 4 1501ctcctttctgggtgttagtt 70 194 1 330002 3′ UTR 4 1511 aacatcatttctcctttctg41 195 1 330003 3′ UTR 4 1536 agctcttgccttatgagttt 58 196 1 330004 3′UTR 4 1546 cttccttctcagctcttgcc 57 197 1 330005 3′ UTR 4 1556aagatcagcgcttccttctc 69 198 1 330006 3′ UTR 4 1566 aattaaatagaagatcagcg57 199 1 330007 3′ UTR 4 1621 gaagagccctgtgaatgtgg 24 200 1 330008 3′UTR 4 1641 agtgtcctgattctgagact 53 201 1 330009 3′ UTR 4 1661cccaaaccagacacctggcc 75 202 1 330010 3′ UTR 4 1681 atgatgatgagcactctgga59 203 1 330011 3′ UTR 4 1691 gttctatgacatgatgatga 59 204 1 330012 3′UTR 4 1719 tcccatttcaggagacctgg 60 205 1 330013 3′ UTR 4 1729ttgctgggcttcccatttca 39 206 1 330014 3′ UTR 4 1739 ctgcgtggtattgctgggct64 207 1 330015 3′ UTR 4 1749 agtggagggactgcgtggta 60 208 1 330016 3′UTR 4 1761 gtgctttgagaaagtggagg 69 209 1 330017 3′ UTR 4 1781attctaatggcctttccagt 61 210 1 330018 3′ UTR 4 1805 aagcagatctgctctgctgg52 211 1 330019 3′ UTR 4 1840 atttatacctagtgcttcat 53 212 1 330020 3′UTR 4 1850 gtaacaacatatttatacct 15 213 1 330021 3′ UTR 4 1860gttcttggcagtaacaacat 74 214 1 330022 3′ UTR 4 1870 agtcatttaagttcttggca67 215 1 330023 3′ UTR 4 1923 agtttcccagaagagccata 45 216 1 330024 3′UTR 4 1952 cccacaaggttcgtgtggaa 53 217 1 330025 3′ UTR 4 1962aattcacagccccacaaggt 2 218 1 330026 3′ UTR 4 1972 atgaagaaagaattcacagc29 219 1 330027 3′ UTR 4 2003 cttgtggcctgggtatattg 59 220 1 330028 3′UTR 4 2013 cacgtccactcttgtggcct 69 221 1 330029 3′ UTR 4 2023ccctgtggttcacgtccact 63 222 1 330030 3′ UTR 4 2033 tgacaggacaccctgtggtt29 223 1 330031 3′ UTR 4 2043 tgggctcctctgacaggaca 66 224 1 330032 3′UTR 4 2043 tcctccagatagggagctgg N.D. 225 330033 3′ UTR 4 2085tatccaactatcctccagat 31 226 1 330034 3′ UTR 4 2095 aacacgtaactatccaacta27 227 1 330035 3′ UTR 4 2105 tcctgctaggaacacgtaac 72 228 1 330036 3′UTR 4 2115 ctgtagttggtcctgctagg 56 229 1 330037 3′ UTR 4 2126ccttgggaagactgtagttg 34 230 1 330038 3′ UTR 4 2136 ataactcaatccttgggaag27 231 1 330039 3′ UTR 4 2146 cccaaagtccataactcaat 22 232 1 330040 3′UTR 4 2156 atgtctcactcccaaagtcc 36 233 1 330041 3′ UTR 4 2166cagcaagaagatgtctcact 50 234 1 330042 3′ UTR 4 2176 ggaaatccagcagcaagaag48 235 1 330043 3′ UTR 4 2186 ctctcagcttggaaatccag 57 236 1 330044 3′UTR 4 2196 ggttcacgtcctctcagctt 76 237 1 330045 3′ UTR 4 2205gtggtcccaggttcacgtcc 50 238 1 330046 3′ UTR 4 2215 atggctactggtggtcccagN.D. 239 330047 3′ UTR 4 2225 ggcaaacaagatggctactg 56 240 1 330048 3′UTR 4 2235 ctctccatgtggcaaacaag 53 241 1 330049 3′ UTR 4 2245ctcacagtctctctccatgt 58 242 1 330050 3′ UTR 4 2255 ggcttctgtcctcacagtct50 243 1 330051 3′ UTR 4 2265 cttccagtttggcttctgtc 65 244 1 330052 3′UTR 4 2275 ggctcctccacttccagttt 71 245 1 330053 3′ UTR 4 2285tcaatcccttggctcctcca 53 246 1 330054 3′ UTR 4 2295 ctgttgtttgtcaatccctt61 247 1 330055 3′ UTR 4 2305 ggtcaaggctctgttgtttg 30 248 1 330056 3′UTR 4 2315 gactccacgtggtcaaggct 79 249 1 330057 3′ UTR 4 2325ctgattcagagactccacgt 69 250 1 330058 3′ UTR 4 2335 ccagacaaggctgattcaga45 251 1 330059 3′ UTR 4 2345 agatctggttccagacaagg 59 252 1 330060 3′UTR 4 2355 gtccaggtgtagatctggtt 53 253 1 330061 3′ UTR 4 2365gacctgggcagtccaggtgt 38 254 1 330062 3′ UTR 4 2378 ttattggcttatagacctgg56 255 1 330063 3′ UTR 4 2410 acagcttggactcactcaag 30 256 1 330064 3′UTR 4 2432 cttctaaagcaactatcaga 10 257 1 330065 3′ UTR 4 2442ttagtcacaacttctaaagc 13 258 1 330066 3′ UTR 4 2452 catagagaagttagtcacaa22 259 1

As shown in Table 2, SEQ ID NOs 85, 95, 112, 121, 122, 129, 135, 136,137, 145, 147, 149, 150, 154, 157, 159, 161, 162, 164, 166, 167, 170,171, 173, 174, 175, 177, 178, 179, 180, 183, 185, 186, 190, 192, 193,194, 196, 197, 198, 199, 201, 202, 203, 204, 205, 207, 208, 209, 210,211, 212, 214, 215, 217, 220, 221, 222, 224, 228, 229, 234, 236, 237,238, 240, 241, 242, 243, 244, 245, 246, 247, 249, 250, 252, 253 and 255demonstrated at least 50% inhibition of human C-reactive proteinexpression in this assay and are therefore preferred. The target regionsto which these preferred sequences are complementary are herein referredto as “preferred target segments” and are therefore preferred fortargeting by compounds of the present invention. These preferred targetsegments are shown in Table 4. These sequences are shown to containthymine (T) but one of skill in the art will appreciate that thymine (T)is generally replaced by uracil (U) in RNA sequences. The sequencesrepresent the reverse complement of the preferred antisense compoundsshown in Table 2. “Target site” indicates the first (5′-most) nucleotidenumber on the particular target nucleic acid to which theoligonucleotide binds. Also shown in Table 4 is the species in whicheach of the preferred target segments was found.

Example 16 Antisense Inhibition of Rat C-Reactive Protein Expression byChimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap

In accordance with the present invention, a series of antisensecompounds was designed to target different regions of the rat C-reactiveprotein RNA, using published sequences (GENBANK® accession numberM83176.1, incorporated herein as SEQ ID NO: 11). The compounds are shownin Table 3. “Target site” indicates the first (5′-most) nucleotidenumber on the particular target nucleic acid to which the compoundbinds. All compounds in Table 3 are chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings”. The wings arecomposed of 2′-O-methoxyethyl (2′-MOE)nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) throughout theoligonucleotide. All cytidine residues are 5-methylcytidines. Thecompounds were analyzed for their effect on rat C-reactive protein mRNAlevels by quantitative real-time PCR as described in other examplesherein. Data, shown in Table 3, are averages from three experiments inwhich primary rat hepatocytes were treated with 150 nM of the antisenseoligonucleotides of the present invention. If present, “N.D.” indicates“no data”.

TABLE 3 Inhibition of rat C-reactive protein mRNA levelsby chimeric phosphorothioate oligonucleotideshaving 2′-MOE wings and a deoxy gap TARGET SEQ ID TARGET % SEQ ISIS #REGION NO SITE SEQUENCE INHIB ID NO 197163 Start Codon 11 1caccatagtagcttctccat 26 260 197164 Coding 11 21 agcttatcgtgatcagaaga 27261 197165 Coding 11 41 atgaccaaaagcctgagaga 26 262 197166 Coding 11 61gcctgtttagacatgtcttc 57 263 197167 Coding 11 81 acactccgggaaatacgaag 47264 197168 Coding 11 101 ggacacataggcagtagctg 61 265 197169 Coding 11121 ttctttgactctgcttccag 36 266 197170 Coding 11 141cagtgaaggcttccagtggc 56 267 197171 Coding 11 161 agcgtgggcatagagacaca 48268 197172 Coding 11 181 ctgaagcttcggctcacatc 23 269 197173 Coding 11201 tggtagcgtaagagaagatg 26 270 197174 Coding 11 221aatctcgttaaagctcgtct 34 271 197175 Coding 11 261 ctgcaatactaaacccttga 38272 197176 Coding 11 281 cagtatttcaggcccaccta 39 273 197177 Coding 11301 ggaatttctgaagcactgaa 30 274 197178 Coding 11 320gatgtgtgttggtacctcag 21 275 197179 Coding 11 411 caatgtagcccttctgcaga 48276 197180 Coding 11 431 gatgcttgcatttgtcccca 51 277 197181 Coding 11451 tcctgctcctgccccaagat 19 278 197182 Coding 11 471caaagccaccgccatacgag 28 279 197183 Coding 11 491 caccaaagactgattcgcgt 14280 197184 Coding 11 511 ttcacatctccaatgtctcc 35 281 197185 Coding 11531 atagcacaaagtcccacatg 53 282 197186 Coding 11 551tgcattgatctgttctggag 37 283 197187 Coding 11 571 aataccctaccaacatagac 47284 197188 Coding 11 601 agtgcccgccagttcaaaac 40 285 197189 Coding 11621 caccgtgtgtttcatacttc 31 286 197190 Coding 11 641ctgcggcttgataaacacat 21 287 197191 Coding 11 661 cagtcagtcaagggccacag 43288 197192 Coding 11 671 ggactcacaacagtcagtca 35 289

As shown in Table 3, SEQ ID NOs 260, 261, 262, 263, 264, 265, 266, 267,268, 270, 271, 272, 273, 274, 276, 277, 279, 281, 282, 283, 284, 285,286, 288 and 289 demonstrated at least 25% inhibition of rat C-reactiveprotein expression in this experiment and are therefore preferred. Thetarget regions to which these preferred sequences are complementary areherein referred to as “preferred target segments” and are thereforepreferred for targeting by compounds of the present invention. Thesepreferred target segments are shown in Table 4. These sequences areshown to contain thymine (T) but one of skill in the art will appreciatethat thymine (T) is generally replaced by uracil (U) in RNA sequences.The sequences represent the reverse complement of the preferredantisense compounds shown in Tables 1, 2 and 3. “Target site” indicatesthe first (5′-most) nucleotide number on the particular target nucleicacid to which the oligonucleotide binds. Also shown in Table 4 is thespecies in which each of the preferred target segments was found.

TABLE 4 Sequence and position of preferred target segmentsidentified in C-reactive protein. TARGET REV SITE SEQ ID TARGET COMP OFSEQ ID NO SITE SEQUENCE SEQ ID ACTIVE IN ID NO 44586 11 16cccgaagctctgacacctgc 19 H. sapiens 290 44587 11 71 ggcgcaaactcccttactgc20 H. sapiens 291 44588 11 181 ccaaaggagtgaattcaggc 21 H. sapiens 29244589 11 221 gacgtgaccatggagaagct 22 H. sapiens 293 44590 11 281ggccagacaggtaagggcca 23 H. sapiens 294 44592 11 341 ggggtctaagactgcatgaa25 H. sapiens 295 44593 11 551 tgtttttctggctcacagac 26 H. sapiens 29644594 11 701 gtgggtacagtattttctcg 27 H. sapiens 297 44595 11 781agttttacagtgggtgggtc 28 H. sapiens 298 44596 11 871 tcagggatcgtggagttctg29 H. sapiens 299 44597 11 1091 gcgggcccttcagtcctaat 30 H. sapiens 30044598 11 1171 ctgtggccctgaggccagct 31 H. sapiens 301 44599 11 1191gtgggtcctgaaggtacctc 32 H. sapiens 302 44600 11 1361ggtaaagtgtctggtctggg 33 H. sapiens 303 44601 11 1391ctatgctgggaaatggtcca 34 H. sapiens 304 44603 11 1671ctggtttgggtccagagtgc 36 H. sapiens 305 44604 11 1711tgctgggcccaggtctcctg 37 H. sapiens 306 44606 11 1961aaccttgtggggctgtgaat 39 H. sapiens 307 44607 11 2161ttgggagtgagacatcttct 40 H. sapiens 308 44608 11 2291agccaagggattgacaaaca 41 H. sapiens 309 44609 11 2431ttctgatagttgctttagaa 42 H. sapiens 310 53590 11 111 ggaggaggtagctctaaggc43 H. sapiens 311 53589 11 201 ccttgtatcactggcagcag 44 H. sapiens 31253587 11 451 tgtgtaactggagaaggggt 46 H. sapiens 313 53585 11 761ggtctaaggatataggatac 48 H. sapiens 314 53584 11 821 ctgaagtcacagtagctcca49 H. sapiens 315 53583 11 861 ggagtccgcctcagggatcg 50 H. sapiens 31653582 11 901 aagcccagggtgaggaagag 51 H. sapiens 317 53581 11 921tctgaagaagggatacactg 52 H. sapiens 318 53580 11 951 agcaagcatcatcttggggc53 H. sapiens 319 53579 11 1031 gaaatgtgaacatgtgggac 54 H. sapiens 32053578 11 1111 gtcctgaactggcgggcact 55 H. sapiens 321 53577 11 1141gtgcaaggcgaagtgttcac 56 H. sapiens 322 53576 11 1341cctcagcgcctgagaatgga 57 H. sapiens 323 53574 11 1551gagctgagaaggaagcgctg 59 H. sapiens 324 53573 11 1611ggagcattgcccacattcac 60 H. sapiens 325 53572 11 1651tcaggacactggccaggtgt 61 H. sapiens 326 53571 11 1771ctcaaagcacactggaaagg 62 H. sapiens 327 53570 11 1831cagagcaaaatgaagcacta 63 H. sapiens 328 53569 11 1971ggctgtgaattctttcttca 64 H. sapiens 329 53568 11 2041ggtgtcctgtcagaggagcc 65 H. sapiens 330 53567 11 2101gatagttacgtgttcctagc 66 H. sapiens 331 53565 11 2211gaacctgggaccaccagtag 68 H. sapiens 332 53564 11 2271agccaaactggaagtggagg 69 H. sapiens 333 53563 11 2341tcagccttgtctggaaccag 70 H. sapiens 334 53562 11 2402cctgtttacttgagtgagtc 71 H. sapiens 335 246578 11 122ctctaaggcaagagatctgg 85 H. sapiens 336 246588 11 258tgaccagcctctctcatgct 95 H. sapiens 337 246605 11 469gtcagtctgtttctcaatct 112 H. sapiens 338 246614 11 562ctcacagacatgtcgaggaa 121 H. sapiens 339 246615 11 567agacatgtcgaggaaggctt 122 H. sapiens 340 246622 11 655gccttcactgtgtgcctcca 129 H. sapiens 341 246628 11 716tctcgtatgccaccaagaga 135 H. sapiens 342 246629 11 726caccaagagacaagacaatg 136 H. sapiens 343 246630 11 736caagacaatgagattctcat 137 H. sapiens 344 246638 11 816ggttcctgaagtcacagtag 145 H. sapiens 345 246640 11 836ctccagtacacatttgtaca 147 H. sapiens 346 246642 11 856agctgggagtccgcctcagg 149 H. sapiens 347 246643 11 860gggagtccgcctcagggatc 150 H. sapiens 348 246647 11 900gaagcccagggtgaggaaga 154 H. sapiens 349 246650 11 944gggcagaagcaagcatcatc 157 H. sapiens 350 246652 11 967gggcaggagcaggattcctt 159 H. sapiens 351 246654 11 987cggtgggaactttgaaggaa 161 H. sapiens 352 246655 11 1000gaaggaagccagtccctggt 162 H. sapiens 353 246657 11 1020gggagacattggaaatgtga 164 H. sapiens 354 246659 11 1040acatgtgggactttgtgctg 166 H. sapiens 355 246660 11 1050ctttgtgctgtcaccagatg 167 H. sapiens 356 246663 11 1097ccttcagtcctaatgtcctg 170 H. sapiens 357 246664 11 1107taatgtcctgaactggcggg 171 H. sapiens 358 246666 11 1127cactgaagtatgaagtgcaa 173 H. sapiens 359 246667 11 1137tgaagtgcaaggcgaagtgt 174 H. sapiens 360 246668 11 1147ggcgaagtgttcaccaaacc 175 H. sapiens 361 246670 11 1235ccacgtctctgtctctggta 177 H. sapiens 362 246671 11 1245gtctctggtacctcccgctt 178 H. sapiens 363 246672 11 1283ccacgtctctgtctctgggc 179 H. sapiens 364 246673 11 1293gtctctgggcctttgttccc 180 H. sapiens 365 246676 11 1348gcctgagaatggaggtaaag 183 H. sapiens 366 246678 11 1368tgtctggtctgggagctcgt 185 H. sapiens 367 246679 11 1378gggagctcgttaactatgct 186 H. sapiens 368 246683 11 1451tcaagttggacagatcttgg 190 H. sapiens 369 246685 11 1481ttacctcacatagatgagaa 192 H. sapiens 370 246686 11 1491tagatgagaaaactaacacc 193 H. sapiens 371 246687 11 1501aactaacacccagaaaggag 194 H. sapiens 372 246689 11 1536aaactcataaggcaagagct 196 H. sapiens 373 246690 11 1546ggcaagagctgagaaggaag 197 H. sapiens 374 246691 11 1556gagaaggaagcgctgatctt 198 H. sapiens 375 246692 11 1566cgctgatcttctatttaatt 199 H. sapiens 376 246694 11 1641agtctcagaatcaggacact 201 H. sapiens 377 246695 11 1661ggccaggtgtctggtttggg 202 H. sapiens 378 246696 11 1681tccagagtgctcatcatcat 203 H. sapiens 379 246697 11 1691tcatcatcatgtcatagaac 204 H. sapiens 380 246698 11 1719ccaggtctcctgaaatggga 205 H. sapiens 381 246700 11 1739agcccagcaataccacgcag 207 H. sapiens 382 246701 11 1749taccacgcagtccctccact 208 H. sapiens 383 246702 11 1761cctccactttctcaaagcac 209 H. sapiens 384 246703 11 1781actggaaaggccattagaat 210 H. sapiens 385 246704 11 1805ccagcagagcagatctgctt 211 H. sapiens 386 246705 11 1840atgaagcactaggtataaat 212 H. sapiens 387 246707 11 1860atgttgttactgccaagaac 214 H. sapiens 388 246708 11 1870tgccaagaacttaaatgact 215 H. sapiens 389 246710 11 1952ttccacacgaaccttgtggg 217 H. sapiens 390 246713 11 2003caatatacccaggccacaag 220 H. sapiens 391 246714 11 2013aggccacaagagtggacgtg 221 H. sapiens 392 246715 11 2023agtggacgtgaaccacaggg 222 H. sapiens 393 246717 11 2043tgtcctgtcagaggagccca 224 H. sapiens 394 246721 11 2105gttacgtgttcctagcagga 228 H. sapiens 395 246722 11 2115cctagcaggaccaactacag 229 H. sapiens 396 246727 11 2166agtgagacatcttcttgctg 234 H. sapiens 397 246729 11 2186ctggatttccaagctgagag 236 H. sapiens 398 246730 11 2196aagctgagaggacgtgaacc 237 H. sapiens 399 246731 11 2205ggacgtgaacctgggaccac 238 H. sapiens 400 246733 11 2225cagtagccatcttgtttgcc 240 H. sapiens 401 246734 11 2235cttgtttgccacatggagag 241 H. sapiens 402 246735 11 2245acatggagagagactgtgag 242 H. sapiens 403 246736 11 2255agactgtgaggacagaagcc 243 H. sapiens 404 246737 11 2265gacagaagccaaactggaag 244 H. sapiens 405 246738 11 2275aaactggaagtggaggagcc 245 H. sapiens 406 246739 11 2285tggaggagccaagggattga 246 H. sapiens 407 246740 11 2295aagggattgacaaacaacag 247 H. sapiens 408 246742 11 2315agccttgaccacgtggagtc 249 H. sapiens 409 246743 11 2325acgtggagtctctgaatcag 250 H. sapiens 410 246745 11 2345ccttgtctggaaccagatct 252 H. sapiens 411 246746 11 2355aaccagatctacacctggac 253 H. sapiens 412 246748 11 2378ccaggtctataagccaataa 255 H. sapiens 413 115255 252 1atggagaagctactatggtg 260 R. norvegicus 414 115256 252 21tcttctgatcacgataagct 261 R. norvegicus 415 115257 252 41tctctcaggcttttggtcat 262 R. norvegicus 416 115258 252 61gaagacatgtctaaacaggc 263 R. norvegicus 417 115259 252 81cttcgtatttcccggagtgt 264 R. norvegicus 418 115260 252 101cagctactgcctatgtgtcc 265 R. norvegicus 419 115261 252 121ctggaagcagagtcaaagaa 266 R. norvegicus 420 115262 252 141gccactggaagccttcactg 267 R. norvegicus 421 115263 252 161tgtgtctctatgcccacgct 268 R. norvegicus 422 115265 252 201catcttctcttacgctacca 270 R. norvegicus 423 115266 252 221agacgagctttaacgagatt 271 R. norvegicus 424 115267 252 261tcaagggtttagtattgcag 272 R. norvegicus 425 115268 252 281taggtgggcctgaaatactg 273 R. norvegicus 426 115269 252 301ttcagtgcttcagaaattcc 274 R. norvegicus 427 115271 252 411tctgcagaagggctacattg 276 R. norvegicus 428 115272 252 431tggggacaaatgcaagcatc 277 R. norvegicus 429 115274 252 471ctcgtatggcggtggctttg 279 R. norvegicus 430 115276 252 511ggagacattggagatgtgaa 281 R. norvegicus 431 115277 252 531catgtgggactttgtgctat 282 R. norvegicus 432 115278 252 551ctccagaacagatcaatgca 283 R. norvegicus 433 115279 252 571gtctatgttggtagggtatt 284 R. norvegicus 434 115280 252 601gttttgaactggcgggcact 285 R. norvegicus 435 115281 252 621gaagtatgaaacacacggtg 286 R. norvegicus 436 115283 252 661ctgtggcccttgactgactg 288 R. norvegicus 437 115284 252 671Tgactgactgttgtgagtcc 289 R. norvegicus 438

As these “preferred target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisensecompounds of the present invention, one of skill in the art armed withthe knowledge of the present invention will recognize or be able toascertain, using no more than routine experimentation, furtherembodiments of the invention that encompass other compounds thatspecifically hybridize to these preferred target segments andconsequently inhibit the expression of C-reactive protein.

According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, siRNAs,external guide sequence (EGS) oligonucleotides, alternate splicers, andother short oligomeric compounds that hybridize to at least a portion ofthe target nucleic acid.

Example 17 Western Blot Analysis of C-Reactive Protein Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 hours after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to C-reactive protein isused, with a radiolabeled or fluorescently labeled secondary antibodydirected against the primary antibody species. Bands are visualizedusing a PHOSPHORIMAGER™ instrument (Molecular Dynamics, SunnyvaleCalif.).

Example 18 Antisense Inhibition of Rabbit C-Reactive Protein Expressionby Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap

In accordance with the present invention, a series of antisensecompounds was designed to target different regions of the rabbitC-reactive protein RNA, using published sequences (GENBANK® accessionnumber M13497.1, incorporated herein as SEQ ID NO: 439). The compoundsare shown in Table 5. “Target site” indicates the first (5′-most)nucleotide number on the particular target nucleic acid to which thecompound binds. All compounds in Table 5 are chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings”. The wings arecomposed of 2′-O-methoxyethyl (2′-MOE)nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) throughout theoligonucleotide. All cytidine residues are 5-methylcytidines.

The compounds were analyzed for their effect on rabbit C-reactiveprotein mRNA levels by quantitative real-time PCR as described in otherexamples herein. Probes and primers to rabbit C-reactive protein weredesigned to hybridize to a rabbit C-reactive protein sequence, usingpublished sequence information (GENBANK® accession number M13497.1,incorporated herein as SEQ ID NO: 439). For rabbit C-reactive proteinthe PCR primers were: forward primer: GGCGCGAGCTGACATATCA (SEQ ID NO:440) reverse primer: CTTGGCAGAGCTCAGGGC (SEQ ID NO: 441) and the PCRprobe was: FAM-TACGTGGTGAAGTACATGTCAAGCCCCAG-TAMRA (SEQ ID NO: 442)where FAM is the fluorescent reporter dye and TAMRA is the quencher dye.For rabbit GAPDH the PCR primers were: forward primer:TGTTCTAGAGACAGCCGCATCTT (SEQ ID NO: 443) reverse primer:CACCGACCTTCACCATCTTGT (SEQ ID NO: 444) and the PCR probe was: 5′JOE-TTGTGCAGTGCCAGCCTCGTCTCA-TAMRA 3′ (SEQ ID NO: 445) where JOE is thefluorescent reporter dye and TAMRA is the quencher dye. Data, shown inTable 5, are averages from three experiments in which primary rabbithepatocytes were treated with 10 nM of the antisense oligonucleotides ofthe present invention. If present, “N.D.” indicates “no data”.

TABLE 5 Inhibition of rabbit C-reactive protein mRNA levelsby chimeric phosphorothioate oligonucleotides having2′-MOE wings and a deoxy gap Target Seq ID Start % SEQ ISIS # Region NOSite SEQUENCE Inhib ID NO 196123 5′ UTR 439 3 cgtctctggctgaaggctca N.D.446 196124 5′ UTR 439 31 ggctcagaatccactccttt N.D. 447 196125 5′ UTR 43951 gccaccagtgctaccgagca N.D. 448 196126 Start Codon 439 71cttctccatggtcactccct N.D. 449 196127 Coding 439 131 catgcctgcctggtcagacaN.D. 450 196128 Coding 439 181 gacacgtaggaattatctga N.D. 451 196129Coding 439 201 tctttaactgtgcgttgagg N.D. 452 196130 Coding 439 241gtgtagaagtagaggcacac N.D. 453 196131 Coding 439 261 cacgagtcatggacagatcaN.D. 454 196132 Coding 439 341 actatatcctatgtccttgg N.D. 455 196133Coding 439 371 gaatattatttcatctccac N.D. 456 196134 Coding 439 421tcccagcttgcacagaggtg N.D. 457 196135 Coding 439 441 ctgcaatgcctgtgctggacN.D. 458 196136 Coding 439 461 cttcccatctacccagagct N.D. 459 196137Coding 439 491 gcccttcttcagactcttcc N.D. 460 196138 Coding 439 526cccagaataatgcttgcctc N.D. 461 196139 Coding 439 601 atgttcacatttccaatgtcN.D. 462 196140 Coding 439 621 gtgaaagtgcatagtcccac N.D. 463 196141Coding 439 661 ctaaaggtcccaccagcata N.D. 464 196142 3′ UTR 439 771caagaagcaccttcaggatc N.D. 465 196143 3′ UTR 439 811 ggtccacagccagaagtatgN.D. 466 196144 3′ UTR 439 841 tagcaggcattcagtatatg N.D. 467 196145 3′UTR 439 921 caatgtagtccacaagatcc N.D. 468 196146 3′ UTR 439 1111accaatgtcctcttcccagt N.D. 469 196147 3′ UTR 439 1181gtgaatgtgggcaactacct N.D. 470 196148 3′ UTR 439 1201ttctgagagtgaatagccct N.D. 471 196149 3′ UTR 439 1221agtcctagctgatagcctaa N.D. 472 196150 3′ UTR 439 1251agaatgagcactgtgaactc N.D. 473 196151 3′ UTR 439 1371gcaagccttctctctaaggc N.D. 474 196152 3′ UTR 439 1411tgactatacccagatgccac N.D. 475 196153 3′ UTR 439 1561cctgactcttgtggcctgaa N.D. 476 196154 3′ UTR 439 1581taggacagcctgagtctcac N.D. 477 196155 3′ UTR 439 1601gagagatggactactctggt N.D. 478 196156 3′ UTR 439 1621gcaacatacagccatccatg N.D. 479 196157 3′ UTR 439 1641gtctgtaattgctcctgcta N.D. 480 196158 3′ UTR 439 1681acgtcttatccccagagtcc N.D. 481 196159 3′ UTR 439 1751tggtcaacaagatagctgca N.D. 482 196160 3′ UTR 439 1801agctctcagctcttccagct N.D. 483 196161 3′ UTR 439 1821cagattccaccactctgtca N.D. 484 196162 3′ UTR 439 1881caggaagtccaggtatagat N.D. 485 196163 3′ UTR 439 1901agctatattagtcacagacc N.D. 486 196164 3′ UTR 439 1951cctctaatgcaaccatcaga N.D. 487 196165 3′ UTR 439 2011atggtcagtctgagctcaca N.D. 488 196166 3′ UTR 439 2041tgccacggactctcccttgc N.D. 489 196167 3′ UTR 439 2071ccttgcaggagactccagat N.D. 490 196168 3′ UTR 439 2221tgaccatgacagcagatttg N.D. 491 196263 3′ UTR 439 2 gtctctggctgaaggctcagN.D. 492 196264 Coding 439 525 ccagaataatgcttgcctct N.D. 493 2802645′UTR 439 27 cagaatccactcctttggag 66 494 280265 5′UTR 439 61gtcactccctgccaccagtg 74 495 280266 Start Codon 439 81accacagcagcttctccatg 25 496 280267 Coding 439 111 tattagagaagctgaccaag27 497 280268 Coding 439 141 ccttcttgtgcatgcctgcc 74 498 280269 Coding439 221 agtgaaggctttgagtggct 25 499 280270 Coding 439 311gaggatctcgttaaattgtc 50 500 280271 Coding 439 364 atttcatctccacccactga60 501 280272 Coding 439 411 cacagaggtgagttggatcc 59 502 280273 Coding439 431 tgtgctggactcccagcttg 63 503 280274 Coding 439 451acccagagctctgcaatgcc 45 504 280275 Coding 439 495 tgtagcccttcttcagactc46 505 280276 Coding 439 544 aacgaatcctgatcctgccc 70 506 280277 Coding439 641 gacggtattaatctcttctg 70 507 280278 3′UTR 439 773cccaagaagcaccttcagga 92 508 280279 3′UTR 439 851 gctgtttatgtagcaggcat 91509 280280 3′UTR 439 881 ctctggtgttgaagaaggca 86 510 280281 3′UTR 4391041 ctaggcgtcaactttctcat 100 511 280282 3′UTR 439 1071tgacttaaaagtcacttctc 46 512 280283 3′UTR 439 1091 taagtggtgaacctgtcttg72 513 280284 3′UTR 439 1121 tagacagaagaccaatgtcc 79 514 280285 3′UTR439 1171 gcaactaccttctactctct 60 515 280286 3′UTR 439 1211gatagcctaattctgagagt 64 516 280287 3′UTR 439 1291 atcttctatttcagaagact81 517 280288 3′UTR 439 1312 agaatggcacagtattgctg 72 518 280289 3′UTR439 1401 cagatgccacttttgcccag 65 519 280290 3′UTR 439 1447atataagcaagcaaacaccc 86 520 280291 3′UTR 439 1571 tgagtctcaccctgactctt56 521 280292 3′UTR 439 1611 gccatccatggagagatgga 47 522 280293 3′UTR439 1631 gctcctgctagcaacataca 85 523 280294 3′UTR 439 1671cccagagtccacactgaatc 67 524 280295 3′UTR 439 1725 cccaggttcatgccttctaa92 525 280296 3′UTR 439 1771 cttctccatctccctccaca 58 526 280297 3′UTR439 1861 ttggttccatgcaaggctga 39 527 280298 3′UTR 439 1891gtcacagacccaggaagtcc 81 528 280299 3′UTR 439 1919 ttcacccaggtaaccaagag77 529 280300 3′UTR 439 1961 gatagtcagacctctaatgc 73 530 280301 3′UTR439 2031 tctcccttgcaaggacagca 57 531 280302 3′UTR 439 2051gagattagagtgccacggac 68 532 280303 3′UTR 439 2081 cagcaagaatccttgcagga85 533 280304 3′UTR 439 2124 cccacacgaatgactaattg 75 534 280305 3′UTR439 2155 gaataagagcattaagaccc 62 535 280306 3′UTR 439 2211agcagatttgagcttctcaa 22 536 280307 3′UTR 439 2271 gaggagtctgtttctacaac10 537 280308 3′UTR 439 2281 ccttacctttgaggagtctg 11 538 280309 3′UTR439 2285 aagcccttacctttgaggag 8 539

As shown in Table 5, SEQ ID NOs 494, 495, 498, 501, 502, 503, 506, 507,508, 509, 510, 511, 513, 514, 515, 516, 517, 518, 519, 520, 521, 523,524, 525, 526, 528, 529, 530, 531, 532, 533, 534 and 535 demonstrated atleast 25% inhibition of rabbit C-reactive protein expression in thisexperiment and are therefore preferred.

Example 19 Antisense Inhibition of Human C-Reactive Protein Expressionby Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap Dose Response Studies

In a further embodiment of the present invention, five oligonucleotideswere selected for additional dose-response studies. Cytokine-inducedHep3B cells were treated with 50, 100 and 150 nM of ISIS 133712, 133719,133726, 140180 and 140177 and mRNA levels were measured 24 hours afteroligonucleotide treatment as described in other examples herein.Untreated cells served as a control.

Results of these studies are shown in Table 6. Data are averages fromtwo experiments and are expressed as percent inhibition ofcytokine-induced control.

TABLE 6 Inhibition of cytokine-induced human C-reactive protein mRNAexpression in Hep3B cells 24 hours after oligonucleotide treatment %Inhibition Dose of oligonucleotide ISIS # 50 nM 100 nM 150 nM SEQ ID NO133712 60 84 77 22 133719 0 46 76 29 133726 75 85 92 36 140177 31 45 1553 140180 26 59 91 56

As shown in Table 6, ISIS 133712, ISIS 133726 and ISIS 140180 wereeffective at reducing human C-reactive protein mRNA levels in adose-dependent manner and are therefore preferred compounds of thepresent invention.

Example 20 Antisense Inhibition of Rat C-Reactive Protein Expression byChimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap Dose Response Studies

In a further embodiment of the present invention, three oligonucleotideswere selected for additional dose-response studies. Rat primaryhepatocytes were treated with 50, 150 and 300 nM of ISIS 197181, 197178,197183 and 197190. Target mRNA levels were measured at 24 hours postoligonucleotide treatment as described in other examples herein.Untreated cells served as a control.

Results of these studies are shown in Table 7. Data are averages fromthree experiments and are expressed as percent inhibition of control.

TABLE 7 Inhibition of rat C-reactive protein mRNA expression in primaryhepatocytes: dose response % Inhibition Dose, nM ISIS # SEQ ID NO 50 150300 197181 278 38 37 37 197178 275 38 56 65 197183 280 9 73 84 197190287 55 71 85

As shown in Table 7, ISIS 197181, ISIS 197178, ISIS 197183 and ISIS197190 were effective at reducing rat C-reactive protein mRNA levels ina dose-dependent manner and are therefore preferred compounds of thepresent invention.

Example 21 Antisense Inhibition of Rat C-Reactive Protein Expression byChimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap In Vivo Dose Response Studies

In a further embodiment of the present invention, three oligonucleotideswere selected for additional in vivo dose response studies. Three-monthold male Sprague-Dawley rats received subcutaneous injections of salineor 1, 10 or 25 mg/kg of ISIS 197178 (SEQ ID NO: 275), ISIS 197183 (SEQID NO: 280) and ISIS 197190 (SEQ ID NO: 287) twice weekly for 2 weeks.At the end of the treatment period, animals were sacrificed and livertarget mRNA levels were measured by real-time PCR as described in otherexamples herein. Saline treated animals served as a control. Rat liverC-reactive protein mRNA levels were reduced by 5% following a 1 mg/kgdose of 197178 and by 18% following a 10 mg/kg dose of ISIS 197190.

Example 22 Antisense Inhibition of Rabbit C-Reactive Protein Expressionby Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap Dose Response Studies

In a further embodiment of the present invention, four oligonucleotideswere selected for additional dose-response studies. Rabbit primaryhepatocytes were treated with 10, 50, 150 and 300 nM of ISIS 280279,280290, 280298 and 282303. mRNA levels were measured 24 hours afteroligonucleotide treatment as described in other examples herein.Untreated cells served as a control.

Results of these studies are shown in Table 8. Data are averages fromtwo experiments and are expressed as percent inhibition of control.

TABLE 8 Inhibition of rabbit C-reactive protein mRNA expression inrabbit primary hepatocytes: dose response % Inhibition Dose ofoligonucleotide ISIS # SEQ ID NO 10 nM 50 nM 150 nM 300 nM 280279 509 5553 62 35 280290 520 49 77 84 81 280298 528 55 53 62 36 282303 533 40 7680 87

As shown in Table 8, ISIS 280303 and ISIS 280290 were effective atreducing C-reactive protein mRNA levels in a dose-dependent manner andare therefore preferred compounds of the present invention.

Example 23 Antisense Inhibition of C-Reactive Protein Expression (ISIS133726) in Liver Tissue of the Cynomolgus Monkey

In a further embodiment, male Cynomolgus monkeys were treated with ISIS133726 (SEQ ID NO: 36) and levels of C-reactive protein mRNA weremeasured in liver tissue.

Male Cynomolgus monkeys were divided into two treatment groups, controlanimals (n=4; saline treatment only) and treated animals (n=8; treatedwith ISIS 133726). Animals in the treatment group were dosedsubcutaneously twice a week for 4 weeks with 10 mg/kg and 20 mg/kg ofISIS 133726, respectively. Animals in the control group were treatedwith saline only. Three days later, all animals were sacrificed andlivers were taken for analysis of C-reactive protein mRNA. Levels ofmRNA were normalized to those of the saline treated animals. In animalstreated with 10 mg/kg and 20 mg/kg ISIS 133726, C-reactive protein mRNAlevels within liver were reduced by 42% and 69%, respectively.

Levels of the liver enzymes ALT and AST were measured weekly and showedno change, indicating no ongoing toxic effects of the oligonucleotidetreatment.

The results of this study demonstrate a significant reduction in liverC-reactive protein mRNA upon treatment with ISIS 133726.

Example 24 Modulation of Mouse C-Reactive Protein Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a series of antisensecompounds was designed to target different regions of the mouseC-reactive protein RNA, using published sequences (GENBANK® accessionnumber NM_(—)007768.1, incorporated herein as SEQ ID NO: 540). Thecompounds are shown in Table 9. “Target site” indicates the first(5′-most) nucleotide number on the particular target nucleic acid towhich the compound binds. All compounds in Table 9 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-O-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

The compounds were analyzed for their effect on mouse C-reactive proteinmRNA levels by quantitative real-time PCR as described in other examplesherein. Probes and primers to mouse C-reactive protein were designed tohybridize to a mouse C-reactive protein sequence, using publishedsequence information (GENBANK® accession number NM_(—)007768.1,incorporated herein as SEQ ID NO: 540). For mouse C-reactive protein thePCR primers were: forward primer: TGGATTGATGGGAAACCCAA (SEQ ID NO: 541)reverse primer: GCATCTGGCCCCACAGTG (SEQ ID NO: 542) and the PCR probewas: FAM-TGCGGAAAAGTCTGCACAAGGGC-TAMRA (SEQ ID NO: 543) where FAM is thefluorescent reporter dye and TAMRA is the quencher dye. For mouse GAPDHthe PCR primers were: forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO:544) reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 545) and the PCRprobe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 546)where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.Data, shown in Table 9, are from an experiment in which primary mousehepatocytes were treated with 150 nM the antisense oligonucleotides ofthe present invention. The data are presented as percent expressionrelative to control, untreated cells. If present, “N.D.” indicates “nodata”.

TABLE 9 Modulation of mouse C-reactive protein mRNA levelsby chimeric phosphorothioate oligonucleotides having2′-MOE wings and a deoxy gap TARGET SEQ SEQ ID TARGET % ID ISIS # REGIONNO SITE SEQUENCE CONTROL NO 133685 5′ UTR 540 21 TTTGTCTGAAAGATCAAGGA 83547 133686 5′ UTR 540 31 AGGACAGTGTTTTGTCTGAA 55 548 133687 start codon540 71 CTTCTCCATGGCTATGGATG 68 549 133688 start codon 540 81ACCAGAGTAGCTTCTCCATG 131 550 133689 coding 540 221 AGTAAAGGTGTTCAGTGGCT84 551 133690 coding 540 301 TTAGAGTTCTTCTTGGTAGC 36 552 133691 coding540 371 GAATCGTACTTCAGCACCAC 139 553 133692 coding 540 411CACAGATGTGTGTTGGAGCC 128 554 133693 coding 540 441 CTACAATCCCCGTAGCAGAC122 555 133694 coding 540 531 CCTGCCCCAAGATGATGCTT 238 556 133695 coding540 661 CTGAGTGTCCCACCAACATA 183 557 133696 coding 540 711CATCACCCTGTGCTTTATAG 175 558 133697 stop codon 540 741GTCAGGACCACAGCTGCGGC 48 559 133698 stop codon 540 761TTCAGGGTTCACAACAGTAG 67 560 133699 3′ UTR 540 781 AATGTAATCCCAGGAGGTGC44 561 133700 3′ UTR 540 891 GTGCTCTAGTGCTGAGGACC 102 562 133701 3′ UTR540 1091 CTCCTTTCTGTGCATCTATT 70 563 133702 3′ UTR 540 1261AGATGATAGGTATTATGCAT 120 564 133703 3′ UTR 540 1361 CCAGTGTCCAGTCTTCAACA52 565 133704 3′ UTR 540 1381 GGGCCCTCCTGATAGATTAT 87 566 133705 3′ UTR540 1425 GTAATCAGTGGCTGCTGAGA 46 567 133706 3′ UTR 540 1451ACAGAACCCTATATGAAGAG 94 568 133707 3′ UTR 540 1508 AGACCTGCATAATGACACCA34 569 133708 3′ UTR 540 1551 GCACAGTGTAGTCAGTGCTC 50 570 147859 5′ UTR540 1 CAAGGAGTCCTGGAACGCCT 414 571 147860 5′ UTR 540 41CTGGACTAAGAGGACAGTGT 81 572 147861 coding 540 102 AGCTGATCATGATCAGAAGG435 573 147862 coding 540 191 TGCTTCCAGAGACACATAGG 262 574 147863 coding540 241 GTGTAGAAATGGAGACACAC 212 575 147864 coding 540 281ATAAGAGAAGACACTGAAGC 129 576 147865 coding 540 501 CCACAGTGTAGCCCTTGTGCN.D. 577 147866 coding 540 521 GATGATGCTTGCATCTGGCC 148 578 147867coding 540 544 TACGAGTCCTGCTCCTGCCC 106 579 147868 coding 540 571GACTGCTTTGCATCAAAGTC 26 580 147869 coding 540 701 TGCTTTATAGTTCAGTGCCC72 581 147870 3′ UTR 540 801 TAACCCGAGACAAGGGAGAG 95 582 147871 3′ UTR540 841 CAGAACAGACCTACAACATC 89 583 147872 3′ UTR 540 861GAAGTGAAAGGCCATATTCA 91 584 147873 3′ UTR 540 931 TAGTGGGATGCTTATGCTGG275 585 147874 3′ UTR 540 1141 AATACAGCACTCAAGATGAC 212 586 147875 3′UTR 540 1181 ATAGGAAAGGATCTGAAGAG 93 587 147876 3′ UTR 540 1211CATCATGAATTTGAGAGAGA 138 588 147877 3′ UTR 540 1281 AGGTAGATAGATTGATTGAT314 589 147878 3′ UTR 540 1301 CTGATGAATAGATGATAGAT 228 590 147879 3′UTR 540 1321 GTAATCAGTAAGATGGATGA 381 591 147880 3′ UTR 540 1378CCCTCCTGATAGATTATCCA 38 592 147881 3′ UTR 540 1501 CATAATGACACCAATTGACA101 593 147882 3′ UTR 540 1521 GGTTGCCCAAACAAGACCTG 144 594 147883 3′UTR 540 1541 GTCAGTGCTCCATCACTCTA 44 595 147884 3′ UTR 540 1561CTGATTCTGAGCACAGTGTA 233 596

Example 25 Antisense Inhibition of Mouse C-reactive Protein Expressionby Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap Dose Response Studies

In a further embodiment of the present invention, seven oligonucleotideswere selected for additional dose-response studies. Primary mousehepatocytes were treated with 10, 50, 150 and 300 nM of ISIS 133688,133697, 133702, 133708, 147880, 147868, 147883. mRNA levels weremeasured 24 hours after oligonucleotide treatment as described in otherexamples herein. Untreated cells served as a control.

Results of these studies are shown in Table 10. Data are averages fromthree experiments and are expressed as percent inhibition of control.

TABLE 10 Inhibition of mouse C-reactive protein mRNA expression in mouseprimary hepatocytes: dose response % Inhibition Dose of oligonucleotideISIS # SEQ ID NO 10 nM 50 nM 150 nM 300 nM 133688 550 59 75 75 67 133697559 63 63 76 76 133702 564 43 35 45 52 133708 570 72 74 72 72 147868 58059 59 76 80 147880 592 61 69 82 77 147883 595 90 82 91 70

As demonstrated in Table 10, ISIS 113697 and 147868 inhibited C-reactiveprotein expression in a dose-dependent manner.

Example 26 Antisense Inhibition of Rabbit C-Reactive Protein In Vivo

In a further embodiment of the present invention, ISIS 280303 (SEQ IDNO: 533) was tested for its effects on C-reactive proteins in rabbits.Male New Zealand white rabbits were fed a normal diet and receivedsubcutaneous injections of 20 mg/kg ISIS 280303 twice per week for aperiod of three weeks. Saline-injected animals served as a control.Oligonucleotide- and saline-injected groups included 4 animals each. Atthe end of the treatment period, the animals were sacrificed and theliver was isolated for RNA extraction. C-reactive protein mRNA levels inliver were measured by real-time PCR as described by other examplesherein. Relative to the saline control, ISIS 280303 inhibited C-reactiveprotein mRNA expression by 52%.

Example 27 Rabbit Models for Study of Atherosclerotic Plaque Formation

The Watanabe heritable hyperlipidemic (WHHL) strain of rabbit is used asa model for atherosclerotic plaque formation. New Zealand white rabbitson a high-fat diet are also used as a model of atherosclerotic plaqueformation. Treatment of WHHL or high fat fed New Zealand white rabbitswith C-reactive protein antisense compounds is used to test theirpotential as therapeutic or prophylactic treatments for atheroscleroticplaque disease. Rabbits are injected with 5, 10, 29 or 50 mg/kg ofantisense oligonucleotides targeted to C-reactive protein. Animalstreated with saline alone or a control oligonucleotide serve ascontrols. Throughout the treatment, serum samples are collected andevaluated for serum lipids, including cholesterol, LDL-cholesterol,VLDL-cholesterol, HDL-cholesterol and triglycerides, by routine clinicalanalysis. Liver tissue triglyceride content is measured using aTriglyceride GPO Assay (Sigma-Aldrich, St. Louis, Mo.). Liver, kidney,heart, aorta and other tissues are procured and processed forhistological analysis using routine procedures. Liver and kidney tissuesare examined for evidence of basophilic granules and inflammatoryinfiltrates. The aorta is stained using routine procedures, with a dyesuch as Sudan IV, to visualize atherosclerosis. Aorta tissue is alsoembedded in paraffin and sectioned, using routine histologicalprocedures, and the sections are evaluated for the presence of intimallesions.

Example 28 A Mouse Model for Atherosclerotic Plaque Formation HumanC-Reactive Protein Transgenic Mice Lacking the LDL Receptor Gene

The LDL receptor is responsible for clearing C-reactiveprotein-containing LDL particles. Without the LDL receptor, animalscannot effectively clear C-reactive protein-containing LDL particlesfrom the plasma, thus the serum levels of C-reactive protein and LDLcholesterol are markedly elevated. Mice expressing the human C-reactiveprotein transgene (TgN-hApoB +/+) and mice deficient for the LDLreceptor (LDLr −/−) are both used as animal models of atheroscleroticplaque development. When the LDL receptor deficiency genotype iscombined with a human C-reactive protein transgenic genotype (TgN-hApoB+/+; LDLr −/−), atherosclerotic plaques develop rapidly. In accordancewith the present invention, mice of this genetic background are used toinvestigate the ability of compounds to prevent atherosclerosis andplaque formation.

Male TgN-hApoB +/+;LDLr −/− mice are treated twice weekly with 10 or 20mg/kg of C-reactive protein antisense oligonucleotides for 12 weeks.Control groups are treated with saline or control oligonucleotide. Serumtotal cholesterol, HDL-cholesterol, LDL-cholesterol and triglyceridesare measured at 2, 4, 6, 8 and 12 weeks by routine clinical analysisusing an Olympus Clinical Analyzer (Olympus America Inc., Melville,N.Y.). Mouse apolipoprotein mRNA in liver is measured at 12 weeks.

Additionally, a four month study is performed in TgN-hApoB +/+;LDLr −/−mice, with treatment conditions used in the 12 week study. Mice aretreated for 4 months with antisense oligonucleotides targeted toC-reactive protein to evaluate the ability of such compounds to preventatherosclerotic plaque formation. Serum total cholesterol,HDL-cholesterol, LDL-cholesterol and triglycerides are measured at 2, 4,6, 8, 12 and 16 weeks by routine clinical analysis using an OlympusClinical Analyzer (Olympus America Inc., Melville, N.Y.). MouseC-reactive protein mRNA in liver at 16 weeks is measured by real-timePCR. At the end of the 4-month treatment period, additional treated miceare anesthetized and perfused with 10% formalin. The perfused arterialtree is isolated and examined for the presence of atheroscleroticplaques. Sections of the arterial tree are embedded in paraffin andprepared for histological analysis using routine methods.

Example 29 A Mouse Model for Atherosclerotic Plaque FormationB6.129P-Apoe^(tm1Unc) Knockout Mice

B6.129P-ApoE^(tm1Unc) knockout mice (herein referred to as ApoE knockoutmice) obtained from The Jackson Laboratory (Bar Harbor, Me.), arehomozygous for the Apoe^(tm1Unc) mutation and show a marked increase intotal plasma cholesterol levels that are unaffected by age or sex. Theseanimals present with fatty streaks in the proximal aorta at 3 months ofage. These lesions increase with age and progress to lesions with lesslipid but more elongated cells, typical of a more advanced stage ofpre-atherosclerotic lesion.

The mutation in these mice resides in the apolipoprotein E (ApoE) gene.The primary role of the ApoE protein is to transport cholesterol andtriglycerides throughout the body. It stabilizes lipoprotein structure,binds to the low density lipoprotein receptor (LDLR) and relatedproteins, and is present in a subclass of HDLs, providing them theability to bind to LDLR. ApoE is expressed most abundantly in the liverand brain. Female B6.129P-Apoetm1Unc knockout mice (ApoE knockout mice)were used in the following studies to evaluate C-reactive proteinantisense oligonucleotides as potential compounds for preventingatherosclerotic plaque formation.

Female ApoE knockout mice range in age from 5 to 7 weeks and are placedon a normal diet for 2 weeks before study initiation. ApoE knockout miceare then fed ad libitum a 60% fat diet, with 0.15% added cholesterol toinduce dyslipidemia and obesity. Control animals are maintained on ahigh-fat diet with no added cholesterol. After overnight fasting, micefrom each group are dosed intraperitoneally every three days with 5, 25or 50 mg/kg of antisense oligonucleotide targeted to C-reactive protein,for a period of six weeks. Control groups consist of animals injectedwith a control oligonucleotide and animals injected with saline.

During and at the end of the treatment period, glucose levels,cholesterol (total cholesterol, HDL-cholesterol and LDL-cholesterol),triglyceride and liver enzyme levels are measured by routine clinicalanalysis using an Olympus Clinical Analyzer (Olympus America Inc.,Melville, N.Y.). At study termination and forty-eight hours after thefinal injections, animals were sacrificed and evaluated for target mRNAlevels in liver by real-time PCR. At the end of the treatment period,additional treated mice are anesthetized and perfused with 10% formalin.The perfused arterial tree is isolated and examined for the presence ofatherosclerotic plaques. Sections of the arterial tree are embedded inparaffin and prepared for histological analysis using routine methods.

Example 30 Antisense Inhibition of Human C-Reactive Protein mRNAExpression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOEWings and a Deoxy Gap Dose Response Study

In a further embodiment, four oligonucleotides were selected for anadditional dose-response study. Cytokine-induced Hep3B cells, culturedas described herein, were treated with 25, 50, 75 and 150 nM of ISIS329956 (SEQ ID NO: 149), ISIS 330012 (SEQ ID NO: 205), ISIS 330031 (SEQID NO: 224) and ISIS 133726 (SEQ ID NO: 36). 24 hours followingoligonucleotide treatment, human C-reactive protein mRNA levels werequantitated using real-time PCR as described herein. ISIS 113529(CTCTTACTGTGCTGTGGACA; incorporated herein as SEQ ID NO: 597) does nottarget C-reactive protein and served as a control. Cells were treatedwith 150 and 300 nM of ISIS 113529. ISIS 113529 is a chimericoligonucleotide (“gapmer”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-O-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

Levels of C-reactive protein mRNA expression were also measured incytokine-induced cells that were not treated with oligonucleotide(induced) and cells that receive neither cytokine nor oligonucleotidetreatment (basal).

The results of this dose-response study are shown in Table 11. Data areaverages from three experiments. Results were normalized to expressionof C-reactive protein mRNA from cytokine-induced cells. Basal C-reactiveprotein mRNA was 11% of the cytokine-induced expression. Cells treatedwith 150 and 300 nM of ISIS 113529 expressed C-reactive protein mRNA at76 and 84% of the cytokine-induced levels, respectively.

TABLE 11 Inhibition of cytokine-induced human C-reactive protein mRNAexpression in Hep3B cells 24 hours after oligonucleotide treatment %C-reactive protein mRNA expression relative to cytokine-induced cellsDose of oligonucleotide ISIS # 25 nM 50 nM 75 nM 150 nM 329956 45 41 2119 330012 48 33 22 12 330031 53 29 21 26 133726 94 51 33 23

These data reveal that ISIS 329956, ISIS 330012, ISIS 330031 and ISIS133726 inhibited human C-reactive protein expression in cytokine-inducedHep3B cells, in a dose-dependent manner.

Example 31 Antisense Inhibition of Human C-Reactive Protein Secretion byHep3B Cells Dose Response Study

In a further embodiment of the present invention, four oligonucleotideswere selected for an additional dose-response study to measure theeffect of antisense oligonucleotide treatment on the secretion ofC-reactive protein from cytokine-induced Hep3B cells. Cytokine-inducedHep3B cells, cultured as described herein, were treated with 150 and 300nM of ISIS 329956 (SEQ ID NO: 149), ISIS 330012 (SEQ ID NO: 205), ISIS330031 (SEQ ID NO: 224) and ISIS 133726 (SEQ ID NO: 36). Cells weretreated with the control oligonucleotide ISIS 113529 (SEQ ID NO: 597) at150 and 300 nM. 24 hours following oligonucleotide treatment humanC-reactive protein secreted from cytokine-induced Hep3B cells into theculture media was measured by ELISA using a commercially available kit(ALerCHEK Inc., Portland, Me.). C-reactive protein secretion was alsomeasured in cytokine-induced cells that were not treated witholigonucleotide (induced) and cells that received neither cytokine noroligonucleotide treatment (basal).

The results of this dose-respose study are shown in Table 12. Data areaverages from three experiments. Results were normalized to C-reactiveprotein levels secreted from cytokine-induced cells. Basal C-reactiveprotein level in the culture media was 8% of the cytokine-induced level.

TABLE 12 Inhibition of cytokine-induced human C-reactive proteinsecretion from Hep3B cells 24 hours after oligonucleotide treatment %C-reactive protein secretion relative to cytokine-induced cells Dose ofoligonucleotide 150 nM 300 nM 329956 71 65 330012 69 47 330031 78 107133726 76 55 113529 127 113

These data reveal that ISIS 329956, ISIS 330012 and ISIS 133726inhibited secretion of C-reactive protein from cytokine-induced Hep3Bcells, in a dose-dependent manner. ISIS 330031 inhibited C-reactiveprotein secretion at the lower dose of oligonucleotide. The controloligonucleotide ISIS 113529 did not inhibit C-reactive proteinsecretion.

Example 32 Antisense Oligonucleotides Targeted to C-Reactive ProteinHaving Variable 2′-Deoxy Gaps and Variable 2′-MOE Wings

In a further embodiment, antisense oligonucleotides targeted toC-reactive protein were designed using the nucleotide sequences of SEQID NOs 36 and 205 and employing various gap and wing segment lengths.The compounds are shown in Table 13. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe compound binds. All compounds in Table 13 are chimericoligonucleotides (“gapmers”) ranging from 16 to 20 nucleotides inlength. The “gap” region consists of 2′-deoxynucleotides, which isflanked on one or both sides (5′ and 3′ directions) by “wings” composedof 2′-O-methoxyethyl (2′-MOE) nucleotides. The length of the 2′-deoxygap varies from 10 to 18 nucleotides and the length of the 2′-MOE wingsvaries from 1 to 5 nucleotides. The exact structure of eacholigonucleotide is designated in Table 13 as the “configuration”. Adesignation of 3˜14˜3, for instance, indicates that the first (5′-most)3 nucleotides and the last (3′-most) 3 nucleotides are 2′-MOEnucleotides and the 14 nucleotides in the gap are 2′-deoxynucleotides.The internucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

TABLE 13 Antisense oligonucleotides targeted to C-reactiveprotein having varying 2′-deoxy gaps and varying 2′-MOE wings TARGETSEQ ID TARGET SEQ ID ISIS # REGION NO SITE SEQUENCE Configuration NO353490 3′ UTR 4 1671 GCACTCTGGACCCAAACCAG 4~12~4 36 353491 3′ UTR 4 1671GCACTCTGGACCCAAACCAG 3~14~3 36 353492 3′ UTR 4 1671 GCACTCTGGACCCAAACCAG2~16~2 36 353470 3′ UTR 4 1719 TCCCATTTCAGGAGACCTGG 4~12~4 205 353471 3′UTR 4 1719 TCCCATTTCAGGAGACCTGG 3~16~1 205 353472 3′ UTR 4 1719TCCCATTTCAGGAGACCTGG 2~16~2 205 353512 3′ UTR 4 1719TCCCATTTCAGGAGACCTGG 3~14~3 205 353480 3′ UTR 4 1719 TCCCATTTCAGGAGACCTG5~10~4 598 353486 3′ UTR 4 1719 CCCATTTCAGGAGACCTGG 4~10~4 599 353499 3′UTR 4 1672 GCACTCTGGACCCAAACCA 5~10~4 600 353502 3′ UTR 4 1671CACTCTGGACCCAAACCAG 4~10~5 601 353481 3′ UTR 4 1720 TCCCATTTCAGGAGACCT5~10~3 602 353483 3′ UTR 4 1721 CCCATTTCAGGAGACCTG 4~10~4 603 353487 3′UTR 4 1719 CCATTTCAGGAGACCTGG 3~10~5 604 353500 3′ UTR 4 1672GCACTCTGGACCCAAACC 5~10~3 605 353503 3′ UTR 4 1671 ACTCTGGACCCAAACCAG3~10~5 606 353505 3′ UTR 4 1673 CACTCTGGACCCAAACCA 4~10~4 607 353484 3′UTR 4 1722 CCATTTCAGGAGACCT 3~10~3 608 353506 3′ UTR 4 1674ACTCTGGACCCAAACC 3~10~3 609

Additional oligonucleotides were designed, using the nucleotide sequenceof SEQ ID Nos 36 and 205 and incorporating uniformly modifiednucleotides. ISIS 353489 and ISIS 353473 (sequences incorporated hereinas SEQ ID Nos 36 and 205, respectively) hybridize to target sites 1671and 1719 of SEQ ID NO: 4, respectively. These two compounds areuniformly comprised of 2′-O-methoxyethyl (2′-MOE) nucleotides, withphosphorothioate internucleoside linkages throughout theoligonucleotide. All cytosines are 5-methylcytosines.

A subset of these antisense oligonucleotides was selected for testing incytokine-induced Hep3B cells. All oligonucleotides tested share the samenucleotide sequence represented herein as SEQ ID NO: 205, and vary withrespect to modifications of the sugar moieties. Cells were cultured andinduced as described herein, and subsequently treated with 50, 100 and200 nM of ISIS 353470, ISIS 353512, ISIS 353472, ISIS 353473 and ISIS330012 for a period of 24 hours. Cytokine-induced cells served as thecontrol to which data were normalized. C-reactive protein mRNA wasmeasured by real-time PCR as described herein. Data, shown in Table 14,represent the average of 3 experiments and are normalized to data fromcells receiving cytokine treatment only. For the gapmers, theconfiguration of each oligonucleotide is indicated in the same manner asdescribed for Table 13. The oligonucleotide uniformly comprised of2′-MOE nucleotides is indicated by “uniform 2′-MOE”.

TABLE 14 Comparison of antisense inhibition by oligonucleotides targetedto C-reactive protein having varying 2′-deoxy gaps and varying 2′-MOEwings % mRNA expression relative to cytokine-induced control cells Doseof oligonucleotide ISIS # Configuration 50 nM 100 nM 200 nM 3534704~12~4 37 28 15 353512 3~14~3 20 16 28 353472 2~16~2 74 42 9 353473Uniform 2′-MOE 117 89 80 330012 5~10~5 55 39 29

Additional oligonucleotides were designed, using the nucleotide sequenceof SEQ ID Nos 36 and 205 and employing differing internucleosidelinkages in the compound. ISIS 353514 and ISIS 353515 (sequencesincorporated herein as SEQ ID Nos 36 and 205, respectively) hybridize totarget sites 1671 and 1719 of SEQ ID NO: 4, respectively. These twocompounds are chimeric oligonucleotides, having a 14 nucleotide gapsegment composed of 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′) by 3 nucleotide wing segments composed of 2′-O-methoxyethyl(2′-MOE) nucleotides. The internucleoside linkages between nucleotides 2and 3 and between nucleotides 18 and 19 are phosphodiester. All othernucleoside linkages in the compounds are phosphorthioate. All cytosinesare 5-methylcytosines.

Additional olignucleotides were designed using the publicly availablesequence of human C-reactive protein (incorporated herein as SEQ ID NO:4). The compounds are shown in Table 15. “Target site” indicates thefirst (5′-most) nucleotide number on the particular target sequence towhich the compound binds. These compounds are hemimers, or “open end”type compounds, 15 nucleotides in length, wherein the “gap” segment islocated at either the 3′ or the 5′ terminus of the oligomeric compoundand consists of 2′-deoxynucleotides. The remaining segment is composedof 2′-O-methoxyethyl (2′-MOE) nucleotides. The exact structure of eacholigonucleotide is designated in Table 15 as the “configuration”. Adesignation of 5-10, for instance, indicates that a 5 nucleotide segmentof a first chemical modification is at the 5′ terminus and a 10nucleotide segment of a second chemical modification is at the 3′terminus. A designation of 2′-MOE-2′-deoxy indicates that the 5′terminus is comprised of 2′-MOE nucleotides, and the 3′ terminus iscomprised of 2′-deoxynucleotides; 2′-MOE nucleotides are furtherindicated in bold type. Where present, “O” indicates that theinternucleoside (backbone) linkages are phosphodiester. All otherinternucleoside linkages are phosphorothioate (P═S). All cytidineresidues are 5-methylcytidines.

TABLE 15 Chimeric hemimers targeted to C-reactive protein TARGET SEQ IDTARGET SEQ ISIS # REGION NO SITE SEQUENCE Configuration ID NO 353698 3′UTR 4 1720 TCCCA _(o)TTTCAGGAGA 5~10 610 2′-MOE~2′-deoxy 353699 3′ UTR 41719 TTTCAGGAGA_(o) CCTGG 10~5 611 2′-deoxy~2′-MOE 353501 3′ UTR 4 1672GCACTCTGGACCCAA 5~10 612 2′-MOE~2′-deoxy 353504 3′ UTR 4 1671CTGGACCCAAACCAG 1~14 613 2′-deoxy~2′-MOE

Example 33 Antisense Inhibition of Human C-Reactive Protein Expressionby Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap Dose Response Studies

In a further embodiment, oligonucleotides targeted to human C-reactiveprotein were selected for additional dose-response studies. Followingantisense oligonucleotide treatment, C-reactive protein mRNA andsecreted protein were measured in primary human hepatocytes, cultured asdescribed herein and cytokine-induced as described herein for Hep3Bcells.

Primary human hepatocytes were treated with 12.5, 25, 50, 100 and 200 nMof ISIS 330012 (SEQ ID NO: 205) and ISIS 133726 (SEQ ID NO: 36).Cytokine-induced cells that did not receive oligonucleotide treatmentserved as controls to which all data were normalized. ISIS 13650(TCCCGCCTGTGACATGCATT, SEQ ID NO: 614) and ISIS 113529 (SEQ ID NO: 597),neither of which target C-reactive protein, served as controloligonucleotides. Cells were treated with 100 and 200 nM of ISIS 113529and ISIS 13650. ISIS 13650 is a chimeric oligonucleotide (“gapmer”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-O-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines.

C-reactive protein mRNA levels were measured after 24 hours ofoligonucleotide treatment by real-time PCR as described in otherexamples herein. Results of these studies are shown in Table 16. Dataare averages from three experiments and are expressed as percent mRNAexpression relative to data from cytokine-induced cells. Where present,“N.D.” indicates not determined.

TABLE 16 Inhibition of human C-reactive protein mRNA expression in humanprimary hepatocytes: 24 hr dose response % mRNA expression relative tocytokine- induced control cells Dose of oligonucleotide ISIS # SEQ ID NO12.5 nM 25 nM 50 nM 100 nM 200 nM 330012 205 42 66 43 45 26 133726 36 5373 56 36 34 113529 597 N.D. N.D. N.D. 73 97 13650 614 N.D N.D. N.D. 7457

As demonstrated in Table 16, doses of 25, 50, 100 and 200 nM of ISIS330012 and 133726 inhibited C-reactive mRNA expression in adose-dependent manner following 24 hours of oligonucleotide treatment.

In a further embodiment, in the same experiment presented in Table 16,C-reactive protein secreted into the tissue culture media from thecytokine-induced primary human hepatocytes was measured by ELISA using acommercially available kit (ALerCHEK Inc., Portland, Me.) following 24hours of oligonucleotide treatment. Data, shown in Table 17, areaverages from three experiments and are expressed as percent proteinsecreted relative to cytokine-induced controls. Where present, “N.D.”indicates not determined.

TABLE 17 Inhibition of human C-reactive protein secretion in humanprimary hepatocytes: 24 hour dose response % Protein secretion relativeto cytokine-induced control cells Dose of oligonucleotide ISIS # SEQ IDNO 12.5 nM 25 nM 50 nM 100 nM 200 nM 330012 205 85 67 61 66 65 133726 3663 67 66 61 68 113529 597 N.D. N.D. N.D. 80 80 13650 614 N.D N.D. N.D.79 91

As demonstrated in Table 17, ISIS 330012 inhibited C-reactive proteinsecretion following 24 hours of oligonucleotide treatment.

In a further embodiment, C-reactive protein mRNA levels incytokine-induced primary human hepatocytes were measured following 48hours of oligonucleotide treatment. Cells were treated with 12.5, 25,50, 100 and 200 nM of ISIS 330012 and ISIS 133726. ISIS 13650 and ISIS113529 served as control oligonucleotides. Cells were treated with 100and 200 nM of ISIS 113529 and ISIS 13650. Data, shown in Table 18, areaverages from three experiments and are expressed as percent mRNAexpression relative to cytokine-induced control cells. Where present,“N.D.” indicates not determined.

TABLE 18 Inhibition of human C-reactive mRNA expression in human primaryhepatocytes: 48 hour dose response % mRNA expression relative tocytokine- induced control cells Dose of oligonucleotide ISIS # SEQ ID NO12.5 nM 25 nM 50 nM 100 nM 200 nM 330012 205 73 53 58 27 19 133726 36 6553 39 34 19 113529 597 N.D. N.D. N.D. 116 79 13650 598 N.D N.D. N.D. 11685

As demonstrated in Table 18, ISIS 330012 and 133726 inhibited C-reactivemRNA expression in a dose-dependent manner following 48 hours ofoligonucleotide treatment.

In a further embodiment, treatment with ISIS 330012 and ISIS 133726 for48 hours was repeated, and both C-reactive protein mRNA and protein weremeasured. C-reactive protein was measured by real-time PCR following 48hours of oligonucleotide treatment. Data, shown in Table 19, areaverages from three experiments and are expressed as percent mRNAexpression relative to cytokine-induced control cells. Where present,“N.D.” indicates not determined.

TABLE 19 Inhibition of human C-reactive protein mRNA expression in humanprimary hepatocytes: 48 hour dose response % mRNA expression relative tocytokine- induced control cells Dose of oligonucleotide ISIS # SEQ ID NO50 100 200 330012 205 54 36 17 133726 36 72 33 25 113529 597 N.D. N.D.112

As demonstrated in Table 19, ISIS 330012 and 133726 inhibited C-reactivemRNA expression in a dose-dependent manner following 48 hours ofoligonucleotide treatment.

In a further embodiment, in the same experiment presented in Table 19,C-reactive protein secreted into the tissue culture media from thecytokine-induced primary human hepatocytes was measured by ELISA using acommercially available kit (ALerCHEK Inc., Portland, Me.) following 48hours of oligonucleotide treatment. Data, shown in Table 20, areaverages from three experiments and are expressed as percent proteinsecreted relative to cytokine-induced controls. Where present, “N.D.”indicates not determined.

TABLE 20 Inhibition of human C-reactive protein secretion in humanprimary hepatocytes: 48 hour dose response % Protein secretion relativeto cytokine- induced control cells Dose of oligonucleotide ISIS # SEQ IDNO 50 100 200 330012 205 40 25 18 133726 36 37 18 20 113529 597 N.D.N.D. 104

As demonstrated in Table 20, ISIS 330012 and 133726 inhibited C-reactiveprotein expression in a dose-dependent manner following 48 hours ofoligonucleotide treatment. At the 200 nM dose, ISIS 133726 and ISIS330012 were able to lower C-reactive protein mRNA in cytokine-inducedcells to levels below basal expression levels, i.e. levels observed incells not induced with cytokine. Northern and immunoblot analyses alsoconfirmed the reduction in C-reactive protein mRNA and proteinexpression after 48 hours of oligonucleotide treatment.

Example 34 Sequencing of Cynomolgus Monkey (Macaca fascicularis)C-Reactive Protein mRNA

In accordance with the present invention, a portion of the cynomolgusmonkey C-reactive protein mRNA not available in the art was amplifiedand sequenced. Positions 537 to 2201 of the human C-reactive proteinmRNA sequence (GENBANK® accession number M11725.1, incorporated hereinas SEQ ID NO: 4) contain the target segment to which ISIS 133726 andISIS 330012 hybridize. The corresponding segment of Cynomolgus monkeyC-reactive protein mRNA was amplified and sequenced, using a series of 8primer sets designed to the human sequence. Total RNA was purified fromCynomolgus monkey primary hepatocytes (In Vitro Technologies,Gaithersburg, Md.). A reverse transcription was performed to producecDNA and was followed by approximately 40 rounds of PCR amplification.Following gel purification of the Cynomolgus fragments, the forward andreverse sequencing reactions of each product were performed using theRETROGEN™ kit (Invitrogen). This kit was used to create thesingle-stranded cDNA and provided reagents for AMPLITAQ™ PCR reaction.The sequenced products were assembled to largely complete the Cynomolgusmonkey C-reactive protein mRNA. This Cynomolgus monkey sequence isincorporated herein as SEQ ID NO: 615 and is 93% homologous to positions537 to 2201 of the human C-reactive protein mRNA. An additional sequencethat shares 97% homology with human C-reactive protein from positions101-290 is incorporated herein as SEQ ID NO: 616.

Example 35 Antisense Inhibition of Cynomolgus Monkey C-Reactive ProteinExpression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap Dose Response Studies

In a further embodiment, oligonucleotides targeted to human C-reactiveprotein were selected for additional dose-response studies were testedfor their ability to inhibit target mRNA in primary Cynomolgus monkeyhepatocytes. Due to the high degree of identity between human andCynmolgus monkey C-reactive protein, ISIS 133726 (SEQ ID NO: 36) andISIS 330012 (SEQ ID NO: 205) hybridize to Cynomolgus monkey C-reactiveprotein with perfect complementarity, at target sites 1147 and 1195 ofthe Cynomolgus monkey mRNA disclosed herein (SEQ ID NO: 615),respectively. Primary Cynolmolgus monkey hepatocytes were induced withcytokine as described herein for Hep3B cells and were treated with 50,100 and 200 nM of ISIS 330012 (SEQ ID NO: 205) and ISIS 133726 (SEQ IDNO: 36). ISIS 113529 (SEQ ID NO: 597) served as the controloligonucleotide. Cells were treated with 150 and 300 nM of ISIS 113529.

C-reactive protein mRNA levels were measured following 24 hours ofoligonucleotide treatment. Data, shown in Table 21, are averages fromthree experiments and are expressed as percent mRNA expression relativeto cytokine-induced controls. Where present, “N.D.” indicates notdetermined.

TABLE 21 Inhibition of Cynomolgus monkey C-reactive protein mRNAexpression in human primary hepatocytes: 24 hour dose response % mRNAxpression elative to cytokine-induced control cells Dose ofoligonucleotide ISIS # SEQ ID NO 25 nM 50 nM 150 nM 300 nM 330012 205 6662 48 13 133726 36 104 111 47 22 113529 597 N.D. N.D. 130 86

As demonstrated in Table 21, ISIS 330012 (at all doses tested) and ISIS133726 (at 150 and 300 nM) inhibited C-reactive protein mRNA expressionin a dose-dependent manner following 24 hours of oligonucleotidetreatment.

In a further embodiment, in the same experiment presented in Table 21,C-reactive protein secreted into the tissue culture media from thecytokine-induced primary Cynomolgus hepatocytes was measured by ELISAusing a commercially available kit (ALerCHEK Inc., Portland, Me.)following 24 hours of oligonucleotide treatment. Data, shown in Table22, are averages from three experiments and are expressed as percentprotein secreted relative to cytokine-induced control cells. Wherepresent, “N.D.” indicates not determined.

TABLE 22 Inhibition of Cynomolgus monkey C-reactive protein secretion inCynomolgus monkey primary hepatocytes: 24 hour dose response % proteinsecretion relative to cytokine induced control cells Dose ofoligonucleotide ISIS # SEQ ID NO 50 100 200 330012 205 40 25 18 13372636 37 18 20 113529 597 N.D. N.D. 104

As demonstrated in Table 22, ISIS 330012 and 133726 inhibited C-reactiveprotein secretion in a dose-dependent manner following 48 hours ofoligonucleotide treatment.

These data demonstrate that ISIS 133726 and ISIS 330012, while designedto target the human C-reactive protein mRNA, are capable of inhibitingboth C-reactive protein mRNA and secreted protein in Cynomolgus monkeyprimary hepatocytes, and are therefore antisense oligonucleotides thatcan be used to test the inhibition of Cynomolgus monkey C-reactiveprotein in vivo.

Example 36 Antisense Inhibition of C-Reactive Protein In Vivo CynomolgusMonkeys

Cynomolgus monkeys (male or female) are useful to evaluate antisenseoligonucleotides for their potential to lower C-reactive protein mRNA orprotein levels, as well as phenotypic endpoints associated withC-reactive protein including, but not limited to cardiovascularindicators, atherosclerosis, lipid diseases, obesity, and plaqueformation. One study includes normal and induced hypercholesterolemicmonkeys fed diets that are normal or high in lipid and cholesterol.Parameters that are observed during the test period include: totalplasma cholesterol, LDL-cholesterol, HDL-cholesterol, triglyceride,arterial wall cholesterol content, and coronary intimal thickening.

In a further embodiment, Cynomolgus monkeys fed an atherogenic dietdevelop atherosclerosis with many similarities to athoroscicroois ofhumans and are used to evaluate the potential of antisense compounds toprevent or ameliorate atherosclerosis. Female Cynomolgus macaques shareseveral similarities in lipoproteins and the cardiovascular system withhumans. In addition to these characteristics, there are similarities inreproductive biology. The Cynomolgus female has a 28-day menstrual cyclelike that of women. Plasma hormone concentrations have been measuredthroughout the Cynomolgus menstrual cycle, and the duration of thefollicular and luteal phases, as well as plasma estradiol andprogesterone concentrations across the cycle, are also remarkablysimilar to those in women.

Antisense oligonucleotides targeted to C-reactive protein are evaluatedfor efficacy and toxicity in Cynomolgus monkeys. The oligonucleotideschosen for these studies hybridize to two distinct regions of the 3′ UTRof both human and monkey C-reactive protein mRNA. ISIS 133726 (SEQ IDNO: 36) and ISIS 330012 (SEQ ID NO: 205) are chimeric oligonucleotideswith a 5˜10˜5 configuration, as described herein. ISIS 353512 (SEQ IDNO: 36) and ISIS 353491 (SEQ ID NO: 205) are the same chimericoligonucleotides, respectively, with a 3˜14˜3 configuration, asdescribed herein. Cynomolgus monkeys are treated as described in Table23. Each of the 9 groups presented in Table 23 consists of 5 animals,and the number of males and females in each of these groups isindicated.

TABLE 23 Treatment of Cynomolgus monkeys with oligonucleotides targetedto C-reactive protein: study design Group Number of # TreatmentFemales/Males Dose mg/kg 1 Saline 3/2 2 ISIS 330012 2/3 7 3 ISIS 3300123/2 20 4 ISIS 133726 2/3 7 5 ISIS 133726 3/2 20 6 ISIS 353512 2/3 7 7ISIS 353512 3/2 20 8 ISIS 353491 2/3 7 9 ISIS 353491 3/2 20

All animals are dosed via subcutaneous injection on the study days 1, 3,5, 8, 11, 15, 18, 22, 25 and 29. The first day of dosing is designatedDay 1. The animals are evaluated for changes in general appearance andbehavior, food consumption and body weight. Blood samples are collectedat 1, 2 and 3 week intervals prior to the start of the study, on days 1and 29 just prior to dosing and at 1, 2, 4 and 24 hours after dosing andon days 8, 15 and 22 just prior to dosing. Blood samples are subjectedto clinical pathology evaluations, which include serum chemistry,hematology, coagulation and urinalysis parameters. Serum chemistryparameters analyzed include sodium, potassium, chloride, carbon dioxide,total bilirubin, alkaline phosphatase (ALP), lactate dehydrogenase(LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT),gamma-glutamyltransferase (GGT), calcium, phosphorus, blood ureanitrogen (BUN), creatinine, total protein, albumin, globulin,albumin/globulin ratio, glucose, cholesterol and triglycerides.Hematology parameters include red blood cell (RBC) counts, white bloodcell (WBC) counts, hemoglobin concentration, hematocrit, reticulocytecounts, plasmodium evaluation, mean corpuscular hemoglobin (MCH), meancorpuscular volume (MCV), mean corpuscular hemoglobin concentration(MCHC), platelet counts and blood cell morphology. Coagulationparameters that are evaluated include activated partial thromboplastintime (APTT) and prothromgin time (PT). Urinalysis parameters that areevaluated include color, character, pH, specific gravity, leukocyteesterase, nitrite, urobilinogen, protein, glucose, ketones, bilirubin,occult blood and microscopics. C-reactive protein in serum is measuredusing an immunochemiluminescence assay (ICMA). All clinical parametersare measured using routine procedures known in the art. Additionally, atoxicokinetic analysis is performed to determine the concentration ofC-reactive protein oligonucleotide in serum. Furthermore, serum levelsof cytokines and chemokines, including interleukin-1, interleukin-6,interleukin-8, interferon-gamma, tumor necrosis factor-alpha, monocytechemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α(MIP-1α), macrophage inflammatory protein-1β (MIP-1β), andregulated-on-activation, normal T cell expressed and secreted cytokine(RANTES), are measured to determine the extent of any immune orinflammatory response.

On day 30 of the study, 24 hours after the final dose of saline oroligonucleotide, animals are sacrificed. Final body weights arerecorded, and a gross necropsy examination is conducted to evaluate thecarcass, muscular/skeletal system, all external surfaces and orifices,cranial cavity and external surface of the brain, neck with associatedorgans and tissues, thoraci, abdominal and pelvic cavities withassociated organs and tissues. Urine is collected from the bladder andanalyzed as previously described herein. Kidney, liver, lung, heart andspleen weights are recorded. Cardiovascular, digestive,lymphoid/hematopoietic, urogenital and endocrine tissues are collectedand preserved in 10% neutral-buffered formalin. Tissues collected fromanimals treated with saline and 20 mg/kg oligonucleotide, followingpreservation in 10% neutral-buffered formalin, are embedded in paraffin,sectioned, stained with hematoxylin and eosin and examined forpathological abnormalities. Bone marrow smears are collected formicroscopic examination in cases where bone marrow sections revealchanges or abnormalities. A portion of the liver tissue collected, whichhas not been preserved in formalin, is homogenized in a buffer thatinhibits Rnase activity and is evaluated for C-reactive protein mRNAexpression by real-time PCR as described herein. The parametersevaluated in this study determine the efficacy and toxicity of antisenseoligonucleotides targeted to C-reactive protein.

Example 37 Antisense Oligonucleotides Targeted to Human C-ReactiveProtein In Vivo Lean Mouse Study

In a further embodiment, antisense oligonucleotides targeted to humanC-reactive protein were tested for their effects on serum lipids, serumglucose and indicators of toxicity. Male C57Bl/6 mice (Charles RiverLaboratories, Wilmington, Mass.) were fed a standard rodent diet. Micewere given intraperitoneal injections of 25 and 50 mg/kg of each of thefollowing antisense oligonucleotides: ISIS 133726 (SEQ ID NO: 36), ISIS329956 (SEQ ID NO: 149), ISIS 330012 (SEQ ID NO: 205) and ISIS 330031(SEQ ID NO: 224). Each oligonucleotide-treated group consisted of 5mice. A total of 10 saline-injected animals served as controls.Injections were administered twice weekly for a period of 4 weeks. Atthe end of the treatment period, mice were sacrificed. Body, liver andspleen weights were recorded and exhibited no significant changes.

Serum was collected for routine clinical analysis of ALT, AST,cholesterol (CHOL), glucose (GLUC), HDL-cholesterol (HDL),LDL-cholesterol (LDL), triglycerides (TRIG) and non-esterified freefatty acids (NEFA). These parameters were measured by routine proceduresusing an Olympus Clinical Analyzer (Olympus America Inc., Melville,N.Y.). The data are presented in Table 24.

TABLE 24 Serum chemistry analysis of mice treated with antisenseoligonucleotides targeted to human C-reactive protein Serum parametersDose ALT AST CHOL GLUC HDL TG LDL NEFA Treatment mg/kg IU/L IU/L mg/dLmg/dL mg/dL mg/dL mg/dL mEq/L SALINE 45 86 81 187 63 132 14 1.0 13372625 36 62 85 172 63 158 16 1.2 50 42 64 73 179 54 139 15 1.4 329956 25 3157 98 172 77 117 17 1.5 50 37 60 105 176 82 149 18 1.7 330012 25 34 7189 200 71 123 13 1.5 50 35 59 93 187 75 115 12 1.5 330031 25 36 94 80194 63 131 14 1.5 50 153 443 150 152 83 131 66 1.6

These data reveal that only the 50 mg/kg dose of ISIS 330031 resulted ina significant increase in the liver transaminases ALT and AST,suggesting a hepatotoxic effect at the highest dose of ISIS 330031.Treatment with ISIS 330031 at 50 mg/kg also resulted in an increase incholesterol and LDL-cholesterol. A moderate increase in cholesterol wasobserved in animals treated with ISIS 329956 at 50 mg/kg. Increases innon-esterified free fatty acids were observed in mice treated with alloligonucleotides used in this study.

These data reveal that antisense oligonucleotides targeted to humanC-reactive protein effectively inhibited target expression in lean mice,without producing overt toxicities.

Example 38 Antisense Inhibition of C-Reactive Protein In Vivo Rat Study

In a further embodiment, antisense oligonucleotides targeted toC-reactive protein were tested in an additional animal model. MaleSprague Dawley rats (Charles River Laboratories, Wilmington, Mass.),maintained on a standard rodent diet, received intraperitonealinjections of 75 and 100 mg/kg ISIS 197178 (SEQ ID NO: 275) once perweek for a period of 6 weeks. Saline-injected animals served ascontrols. Each treatment group consisted of 5 animals. At the end of thetreatment period, the animals were sacrificed and evaluated forC-reactive protein mRNA and protein expression and liver, as well asC-reactive protein expression in serum. mRNA was measured by real-timePCR as described by other examples herein. Protein was measured by ELISAusing a commercially available kit (BD Biosciences, Bedford, Mass.). Thedata, averaged from the 5 animals in each treatment group, arenormalized to results from saline-treated animals and are presented inTable 25.

TABLE 25 Effects of antisense inhibition of C-reactive protein in rats %control Dose of ISIS 197178 C-reactive protein: 75 mg/kg 100 mg/kg mRNA12 13 protein, serum 15 15 protein, liver 32 33

These data demonstrate that ISIS 197178 markedly decreased liverC-reactive protein mRNA and protein, as well as serum protein. Reductionof serum C-reactive protein levels was confirmed by immunoblot analysisusing the rat C-reactive protein antibody from the ELISA kit. Theseresults reveal that reduction in liver C-reactive protein mRNA lowersserum C-reactive protein levels, illustrating an important link betweenliver C-reactive protein production and serum levels.

Example 39 Specificity of Oligonucleotides Targeted to C-ReactiveProtein

In a further embodiment, the specificity of ISIS 330012 to C-reactiveprotein mRNA was investigated. A BLAST search was conducted to determinewhether ISIS 330012 could hybridize to genes other than C-reactiveprotein. This search revealed several genes with sequences that harborpotential binding sites for ISIS 330012. These genes are shown in Table26, where the number of mismatches is indicated. All potential ISIS330012 target sites contain 2-3 mismatched nucleotides with respect toISIS 330012. Also shown are the Unigene ID accession numbers ofsequences, both of which are available through the National Center forBiotechnology Information database. The number of times the binding siteis repeated in the gene sequence is indicated in the “count” column inTable 26.

TABLE 26 Gene sequences sharing 2-3 mismatches with C-reactive proteinat the ISIS 330012 binding site # Mis- Unigene GENBANK ® matches IDAccession # Gene Name Count 2 Hs.256184 NM_001404.1 eukaryotictranslation 1 elongation factor 1 gamma 2 Hs.441043 NM_014817.1 importin11 1 2 Hs.54971 NM_016505.1 putative S1 RNA binding 1 domain protein 3Hs.11417 NM_006423.1 Rab acceptor 1 3 (prenylated) 3 Hs.121549NM_145752.1 CDP-diacylglycerol-- 1 inositol 3-phosphatidyltransferase(phosphatidylinositol synthase) 3 Hs.131842 NM_015255.1 ubiquitin ligaseE3 2 alpha-II 3 Hs.135226 NM_001908.1 cathepsin B 1 3 Hs.135805BC016490.1 skeletrophin 1 3 Hs.180577 NM_002087.1 granulin 1 3 Hs.200063NM_015401.1 histone deacetylase 7A 1 3 Hs.20157 NM_025197.1 CDK5regulatory subunit 1 associated protein 3 3 Hs.248017 NM_014364.1glyceraldehyde-3- 1 phosphate dehydrogenase, testis- specific 3Hs.274268 NM_145648.1 solute carrier family 15, 1 member 4 3 Hs.387667AF106698.1 peroxisome proliferative 1 activated receptor, gamma 3Hs.418167 NM_000477.3 albumin 2

To test whether ISIS 330012 affects the expression of the genes in Table26, primary human hepatocytes, cultured as described herein, weretreated with 200 nM ISIS 330012 for 48 hours. Expression of the genes inTable 26 was measured by real-time PCR as described herein, usingprimers and probes designed to publicly available sequences. These datarevealed that ISIS 330012 did not modulate the expression of any of thegenes in Table 26, illustrating that, in primary hepatocytes, ISIS330012 specifically hybridizes to, and inhibits, C-reactive proteinmRNA.

Example 40 Cell Proliferation and Survival in Response to Cells Treatedwith Oligomeric Compounds Targeted to C-Reactive Protein

Cell cycle regulation is the basis for various cancer therapeutics.Unregulated cell proliferation is a characteristic of cancer cells, thusmost current chemotherapy agents target dividing cells, for example, byblocking the synthesis of new DNA required for cell division. However,cells in healthy tissues are also affected by agents that modulate cellproliferation.

In some cases, a cell cycle inhibitor causes apoptosis in cancer cells,but allows normal cells to undergo growth arrest and therefore remainunaffected (Blagosklonny, Bioessays, 1999, 21, 704-709; Chen et al.,Cancer Res., 1997, 57, 2013-2019; Evan and Littlewood, Science, 1998,281, 1317-1322; Lees and Weinberg, Proc. Natl. Acad. Sci. USA, 1999, 96,4221-4223). An example of sensitization to anti-cancer agents isobserved in cells that have reduced or absent expression of the tumorsuppressor genes p53 (Bunz et al., Science, 1998, 282, 1497-1501; Bunzet al., J. Clin. Invest., 1999, 104, 263-269; Stewart et al., CancerRes., 1999, 59, 3831-3837; Wahl et al., Nat. Med., 1996, 2, 72-79).However, cancer cells often escape apoptosis (Lowe and Lin,Carcinogenesis, 2000, 21, 485-495; Reed, Cancer J. Sci. Am., 1998, 4Suppl 1, S8-14). Further disruption of cell cycle checkpoints in cancercells can increase sensitivity to chemotherapy while allowing normalcells to take refuge in G1 and remain unaffected. Cell cycle assays areemployed to identify genes, such as p53, whose inhibition sensitizescells to anti-cancer agents.

Cell Cycle Assay

The effect of oligomeric compounds targeted to C-reactive protein wereexamined in normal human mammary epithelial cells (HMECs) as well as intwo breast carcinoma cell lines, MCF7 and T47D. All of the cell linesare obtained from the American Type Culture Collection (Manassas, Va.).The latter two cell lines express similar genes. MCF7 cells express thetumor suppressor p53, while T47D cells are deficient in p53. MCF-7 andHMECs cells are routinely cultured in DMEM low glucose (Invitrogen LifeTechnologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum(Invitrogen Life Technologies, Carlsbad, Calif.). T47D cells werecultured in DMEM High glucose media (Invitrogen Life Technologies,Carlsbad, Calif.) supplemented with 10% fetal bovine serum. Cells wereroutinely passaged by trypsinization and dilution when they reachedapproximately 90% confluence. Cells were plated in 24-well plates atapproximately 50,000-60,000 cells per well for HMEC cells, approximately140,000 cells per well for MCF-7 and approximately 170,000 cells perwell for T47D cells, and allowed to attach to wells overnight.

ISIS 133726 (SEQ ID NO: 36) was used to test the effects of antisenseinhibition of C-reactive protein on cell cycle progression. A randomizedcontrol oligonucleotide, ISIS 29848 (NNNNNNNNNNNNNNNNNNNN; where N is A,T, C or G; herein incorporated as SEQ ID NO: 617) was used a negativecontrol, a compound that does not modulate cell cycle progression. Inaddition, a positive control for the inhibition of cell proliferationwas assayed. The positive control was ISIS 148715 (TTGTCCCAGTCCCAGGCCTC;herein incorporated as SEQ ID NO: 618), which targets human Jagged2 andis known to inhibit cell cycle progression. ISIS 29248 and ISIS 148715are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length,composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-O-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines.

Oligonucleotide was mixed with LIPOFECTIN™ reagent (Invitrogen LifeTechnologies, Carlsbad, Calif.) in OPTI-M™ medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) to acheive a final concentration of 200nM of oligonucleotide and 6 μg/mL LIPOFECTIN™ reagent. Before adding tocells, the oligonucleotide, LIPOFECTIN™ reagent and OPTI-MEM™ mediumwere mixed thoroughly and incubated for 0.5 hrs. The medium was removedfrom the plates and the plates were tapped on sterile gauze. Each wellcontaining T47D or MCF7 cells was washed with 150 μl ofphosphate-buffered saline. Each well containing HMECs was washed with150 μL of Hank's balanced salt solution. The wash buffer in each wellwas replaced with 100 μL of the oligonucleotide/OPTI-MEM™medium/LIPOFECTIN™ reagent cocktail. Control cells received LIPOFECTIN™reagent only. The plates were incubated for 4 hours at 37° C., afterwhich the medium was removed and the plate was tapped on sterile gauze.100 μl of full growth medium was added to each well. After 72 hours,routine procedures were used to prepare cells for flow cytometryanalysis and cells were stained with propidium iodide to generate a cellcycle profile using a flow cytometer. The cell cycle profile wasanalyzed with the ModFit program (Verity Software House, Inc., TopshamMe.).

Fragmentation of nuclear DNA is a hallmark of apoptosis and produces anincrease in cells with a hypodiploid DNA content, which are categorizedas “subG1”. An increase in cells in G1 phase is indicative of a cellcycle arrest prior to entry into S phase; an increase in cells in Sphase is indicative of cell cycle arrest during DNA synthsis; and anincrease in cells in the G2/M phase is indicative of cell cycle arrestjust prior to or during mitosis. Data are expressed as percentage ofcells in each phase relative to the cell cycle profile of untreatedcontrol cells and are shown in Table 27.

TABLE 27 Cell cycle profile of cells treated with oligomeric compoundstargeted to C-reactive protein G1 S G2/M Treatment Sub G1 Phase PhasePhase HMEC ISIS 133726 135 101 80 111 ISIS 29848 117 99 82 113 ISIS148715 47 99 88 107 MCF7 ISIS 133726 116 110 83 103 ISIS 29848 130 10691 98 ISIS 148715 42 109 80 110 T47D ISIS 133726 349 82 111 130 ISIS29848 154 86 111 118 ISIS 148715 62 83 116 124

These data reveal that ISIS 133726 did not significantly affect cellcycle progression in HMECs, MCF7 cells or T47D cells.

Caspase Assay

Programmed cell death, or apoptosis, is an important aspect of variousbiological processes, including normal cell turnover, as well as immunesystem and embryonic development. Apoptosis involves the activation ofcaspases, a family of intracellular proteases through which a cascade ofevents leads to the cleavage of a select set of proteins. The caspasefamily can be divided into two groups: the initiator caspases, such ascaspase-8 and -9, and the executioner caspases, such as caspase-3, -6and -7, which are activated by the initiator caspases. The caspasefamily contains at least 14 members, with differing substratepreferences (Thornberry and Lazebnik, Science, 1998, 281, 1312-1316). Acaspase assay is utilized to identify genes whose inhibition selectivelycauses apoptosis in breast carcinoma cell lines, without affectingnormal cells, and to identify genes whose inhibition results in celldeath in the p53-deficient T47D cells, and not in the MCF7 cells whichexpress p53 (Ross et al., Nat. Genet., 2000, 24, 227-235; Scherf et al.,Nat. Genet., 2000, 24, 236-244). The chemotherapeutic drugs taxol,cisplatin, etoposide, gemcitabine, camptothecin, aphidicolin and5-fluorouracil all have been shown to induce apoptosis in acaspase-dependent manner.

In a further embodiment of the invention, oligomeric compounds targetedto C-reactive protein were examined in normal human mammary epithelialcells (HMECs) as well as in two breast carcinoma cell lines, MCF7 andT47D. HMECs and MCF7 cells express p53, whereas T47D cells do notexpress this tumor suppressor gene. Cells were cultured as described forthe cell cycle assay in 96-well plates with black sides and flat,transparent bottoms (Corning Incorporated, Corning, N.Y.). DMEM media,with and without phenol red, were obtained from Invitrogen LifeTechnologies (Carlsbad, Calif.). MEGM media, with and without phenolred, were obtained from Cambrex Bioscience (Walkersville, Md.).

ISIS 133726 (SEQ ID NO: 36) was used to test the effects of antisenseinhibition of C-reactive protein on caspase-activity. A randomizedcontrol oligonucleotide, ISIS 29848 (NNNNNNNNNNNNNNNNNNNN; where N is A,T, C or G; incorporated herein as SEQ ID NO: 617) was used as a negativecontrol, a compound that does not effect caspase activity. As a positivecontrol for caspase activation, an oligonucleotide targeted to humanJagged2 ISIS 148715 (SEQ ID NO: 618) or human Notch1 ISIS 226844(GCCCTCCATGCTGGCACAGG; herein incorporated as SEQ ID NO: 619) was alsoassayed. Both of these genes are known to induce caspase activity, andsubsequently apoptosis, when inhibited. ISIS 29248, ISIS 148715 and ISIS226844 are all chimeric oligonucleotides (“gapmers”) 20 nucleotides inlength, composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-O-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines.

Oligonucleotide was mixed with LIPOFECTIN™ reagent (Invitrogen LifeTechnologies, Carlsbad, Calif.) in OPTI-MEM™ medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) to acheive a final concentration of 200nM of oligonucleotide and 6 μg/mL LIPOFECTIN™ reagent. Before adding tocells, the oligonucleotide, LIPOFECTIN™ reagent and OPTI-MEM™ mediumwere mixed thoroughly and incubated for 0.5 hrs. The medium was removedfrom the plates and the plates were tapped on sterile gauze. Each wellwas washed in 150 μl of phosphate-buffered saline (150 μL Hank'sbalanced salt solution for HMEC cells). The wash buffer in each well wasreplaced with 100 μL of the oligonucleotide/OPTI-MEM™ medium/LIPOFECTIN™reagent cocktail. Compounds targeted to C-reactive protein, ISIS 226844and ISIS 148715 were tested in triplicate, and ISIS 29848 was tested inup to six replicate wells. Untreated control cells received LIPOFECTIN™reagent only. The plates were incubated for 4 hours at 37° C., afterwhich the medium was removed and the plate was tapped on sterile gauze.100 μl of full growth medium without phenol red was added to each well.

Caspase-3 activity was evaluated with a fluorometric HTS Caspase-3 assay(Catalog # HTS02; EMD Biosciences, San Diego, Calif.) that detectscleavage after aspartate residues in the peptide sequence (DEVD). TheDEVD substrate is labeled with a fluorescent molecule, which exhibits ablue to green shift in fluorescence upon cleavage by caspase-3. Activecaspase-3 in the oligonucleotide treated cells is measured by this assayaccording to the manufacturer's instructions. 48 hours followingoligonucleotide treatment, 50 uL of assay buffer containing 10 μMdithiothreitol was added to each well, followed by addition 20 uL of thecaspase-3 fluorescent substrate conjugate. Fluorescence in wells wasimmediately detected (excitation/emission 400/505 nm) using afluorescent plate reader (SPECTRAMAX™ GEMINIXS™ reader, MolecularDevices, Sunnyvale, Calif.). The plate was covered and incubated at 37°C. for and additional three hours, after which the fluorescence wasagain measured (excitation/emission 400/505 nm). The value at time zerowas subtracted from the measurement obtained at 3 hours. The measurementobtained from the untreated control cells was designated as 100%activity.

The experiment was replicated in each of the 3 cell types, HMECs, T47Dand MCF7 and the results are shown in Table 28. From these data, valuesfor caspase activity above or below 100% are considered to indicate thatthe compound has the ability to stimulate or inhibit caspase activity,respectively. The data are shown as percent increase in fluorescencerelative to untreated control values.

TABLE 28 Effects of antisense inhibition of C-reactive protein onapoptosis in the caspase assay Percent relative to Cell Type Treatmentuntreated control HMEC ISIS 133726 148 ISIS 29848 275 ISIS 148715 1006MCF7 ISIS 133726 77 ISIS 29848 103 ISIS 226844 199 T47D ISIS 133726 125ISIS 29848 154 ISIS 148715 380

From these data it is evident that inhibition of C-reactive proteinexpression by ISIS 133726 resulted in an inhibition of apoptosis in MCF7cells, as compared to untreated control cells controls. These dataindicate that this oligomeric compound is a candidate agent withapplications in the treatment of conditions in which inhibition ofapoptosis is desirable, for example, in neurodegenerative disorders.

Example 41 Assay for Inhibition of Angiogenesis Using OligomericCompounds Targeted to C-Reactive Protein

Angiogenesis is the growth of new blood vessels (veins and arteries) byendothelial cells. This process is important in the development of anumber of human discascs, and is believed to be particularly importantin regulating the growth of solid tumors. Without new vessel formationit is believed that tumors will not grow beyond a few millimeters insize. In addition to their use as anti-cancer agents, inhibitors ofangiogenesis have potential for the treatment of diabetic retinopathy,cardiovascular disease, rheumatoid arthritis and psoriasis (Carmelietand Jain, Nature, 2000, 407, 249-257; Freedman and Isner, J. Mol. Cell.Cardiol., 2001, 33, 379-393; Jackson et al., Faseb J., 1997, 11,457-465; Saaristo et al., Oncogene, 2000, 19, 6122-6129; Weber and DeBandt, Joint Bone Spine, 2000, 67, 366-383; Yoshida et al., Histol.Histopathol., 1999, 14, 1287-1294).

Endothelial Tube Formation Assay as a Measure of Angiogenesis

Angiogenesis is stimulated by numerous factors that promote interactionof endothelial cells with each other and with extracellular matrixmolecules, resulting in the formation of capillary tubes. Thismorphogenic process is necessary for the delivery of oxygen to nearbytissues and plays an essential role in embryonic development, woundhealing, and tumor growth (Carmeliet and Jain, Nature, 2000, 407,249-257). Moreover, this process can be reproduced in a tissue cultureassay that evaluated the formation of tube-like structures byendothelial cells. There are several different variations of the assaythat use different matrices, such as collagen I (Kanayasu et al.,Lipids, 1991, 26, 271-276), Matrigel (Yamagishi et al., J. Biol. Chem.,1997, 272, 8723-8730) and fibrin (Bach et al., Exp. Cell Res., 1998,238, 324-334), as growth substrates for the cells. In this assay, HUVECsare plated on a matrix derived from the Engelbreth-Holm-Swarm mousetumor, which is very similar to Matrigel (Kleinman et al., Biochemistry,1986, 25, 312-318; Madri and Pratt, J. Histochem. Cytochem., 1986, 34,85-91). Untreated HUVECs form tube-like structures when grown on thissubstrate. Loss of tube formation in vitro has been correlated with theinhibition of angiogenesis in vivo (Carmeliet and Jain, Nature, 2000,407, 249-257; Zhang et al., Cancer Res., 2002, 62, 2034-2042), whichsupports the use of in vitro tube formation as an endpoint forangiogenesis.

In a further embodiment, primary human umbilical vein endothelial cells(HuVECs) were used to measure the effects of oligomeric compoundstargeted to C-reactive protein on tube formation activity. HuVECs wereroutinely cultured in EBM (Clonetics Corporation, Walkersville, Md.)supplemented with SingleQuots supplements (Clonetics Corporation,Walkersville, Md.). Cells were routinely passaged by trypsinization anddilution when they reached approximately 90% confluence and weremaintained for up to 15 passages. HuVECs are plated at approximately3000 cells/well in 96-well plates. One day later, cells are transfectedwith antisense oligonucleotides. The tube formation assay is performedusing an in vitro Angiogenesis Assay Kit (Chemicon International,Temecula, Calif.).

ISIS 133726 (SEQ ID NO: 36) was used to test the effects of inhibitionof C-reactive protein on endothelial tube formation. A randomizedcontrol oligonucleotide, ISIS 29848 (NNNNNNNNNNNNNNNNNNNN; where N is A,T, C or G; herein incorporated as SEQ ID NO: 617) served as a negativecontrol, a compound that does not affect tube formation. ISIS 196103(AGCCCATTGCTGGACATGCA, incorporated herein as SEQ ID NO: 620) which istargeted to integrin-β3 and is known to inhibit endothelial tubeformation, was used as a positive control

Oligonucleotide was mixed with LIPOFECTIN™ reagent (Invitrogen LifeTechnologies, Carlsbad, Calif.) in OPTI-MEM™ medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) to achieve a final concentration of 75nM of oligonucleotide and 2.25 μg/mL LIPOFECTIN™ reagent. Before addingto cells, the oligonucleotide, LIPOFECTIN™ reagent and OPTI-MEM™ mediumwere mixed thoroughly and incubated for 0.5 hrs. Untreated control cellsreceived LIPOFECTIN™ reagent only. The medium was removed from theplates and the plates were tapped on sterile gauze. Each well was washedin 150 μl of phosphate-buffered saline. The wash buffer in each well wasreplaced with 100 μL of the oligonucleotide/OPTI-MEM™ medium/LIPOFECTIN™reagent cocktail. ISIS 133726 and ISIS 196103 were tested in triplicate,and ISIS 29848 was tested in up to six replicates. The plates wereincubated for 4 hours at 37° C., after which the medium was removed andthe plate was tapped on sterile gauze. 100 μl of full growth medium wasadded to each well. Fifty hours after transfection, cells aretransferred to 96-well plates coated with ECMa-trix™ (ChemiconInter-national). Under these conditions, untreated HUVECs form tube-likestructures. After an overnight incubation at 37° C., treated anduntreated cells are inspected by light microscopy. Individual wells areassigned discrete scores from 1 to 5 depending on the extent of tubeformation. A score of 1 refers to a well with no tube formation while ascore of 5 is given to wells where all cells are forming an extensivetubular network. Results are expressed as percent tube formationrelative to untreated control samples. Treatment with ISIS 133726, ISIS29848 and ISIS 196103 resulted in 81%, 100% and 51% tube formation,respectively. These results illustrate that ISIS 133726 inhibited tubeformation and is thus a candidate agent with applications in thetreatment of conditions where the inhibition of angiogenesis isdesirable, for example, in the treatment of cancer, diabeticretinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis.

Matrix Metalloproteinase Activity

During angiogenesis, endothelial cells must degrade the extracellularmatrix (ECM) and thus secrete matrix metalloproteinases (MMPs) in orderto accomplish this degradation. MMPs are a family of zinc-dependentendopeptidases that fall into eight distinct classes: five are secretedand three are membrane-type MMPs (MT-MMPs) (Egeblad and Werb, J. CellScience, 2002, 2, 161-174). MMPs exert their effects by cleaving adiverse group of substrates, which include not only structuralcomponents of the extracellular matrix, but also growth-factor-bindingproteins, growth-factor pre-cursors, receptor tyrosine-kinases,cell-adhesion molecules and other proteinases (Xu et al., J. Cell Biol.,2002, 154, 1069-1080).

In a further embodiment, the antisense inhibition of apolipoprotein Bwas evaluated for effects on MMP activity in the media above humanumbilical-vein endothelial cells (HUVECs). MMP activity was measuredusing the EnzChek Gelatinase/Collagenase Assay Kit (Molecular Probes,Eugene, Oreg.). HUVECs are cultured as described for the tube formationassay. HUVECs are plated at approximately 4000 cells per well in 96-wellplates and transfected one day later.

HUVECs were treated with ISIS 133726 (SEQ ID NO: 36) to inhibitC-reactive protein expression. An oligonucleotide with a randomizedsequence, ISIS 29848 (NNNNNNNNNNNNNNNNNNNN; where N is A, T, C or G;herein incorporated as SEQ ID NO: 617) served as a negative control, ora treatment not expected to affect MMP activity. ISIS 25237(GCCCATTGCTGGACATGC, SEQ ID NO: 621) targets integrin beta 3 and wasused as a positive control for the inhibition of MMP activity. ISIS25237 is a chimeric oligonucleotide (“gapmers”) 18 nucleotides inlength, composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by four-nucleotide “wings”. The wings are composed of2′-O-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotides. Allcytidine residues are 5-methylcytidines.

Cells were treated as described for the tube formation assay, with 75 nMof oligonucleotide and 2.25 μg/mL LIPOFECTIN™ reagent. ISIS 133726 andISIS 25237 were tested in triplicate, and the ISIS 29848 was tested inup to six replicates. The plates were incubated for approximately 4hours at 37° C., after which the medium was removed and the plate wastapped on sterile gauze. 100 μl of full growth medium was added to eachwell. Approximately 50 hours after transfection, a p-aminophenylmercuricacetate (APMA, Sigma-Aldrich, St. Louis, Mo.) solution is added to eachwell of a Corning-Costar 96-well clear bottom plate (VWR International,Brisbane, Calif.). The APMA solution is used to promote cleavage ofinactive MMP precursor proteins. Media above the HUVECs is thentransferred to the wells in the 96-well plate. After 30 minutes, thequenched, fluorogenic MMP cleavage substrate is added, and baselinefluorescence is read immediately at 485 nm excitation/530 nm emission.Following an overnight incubation at 37° C. in the dark, plates are readagain to determine the amount of fluorescence, which corresponds to MMPactivity. Total protein from HUVEC lysates is used to normalize thereadings, and MMP activites are expressed as a percent relative to MMPactivity from untreated control cells that did not receiveoligonucleotide treatment. MMP activities were 78%, 82% and 58% in theculture media from cells treated with ISIS 133726, ISIS 29848 and ISIS25237. These data reveal that ISIS 133726 did not inhibit MMP activity.

Example 42 Adipocyte Assay of Oligomeric Compounds Targeted toC-Reactive Protein

Insulin is an essential signaling molecule throughout the body, but itsmajor target organs are the liver, skeletal muscle and adipose tissue.Insulin is the primary modulator of glucose homeostasis and helpsmaintain a balance of peripheral glucose utilization and hepatic glucoseproduction. The reduced ability of normal circulating concentrations ofinsulin to maintain glucose homeostasis manifests in insulin resistancewhich is often associated with diabetes, central obesity, hypertension,polycystic ovarian syndrom, dyslipidemia and atherosclerosis (Saltiel,Cell, 2001, 104, 517-529; Saltiel and Kahn, Nature, 2001, 414, 799-806).

Response of Undifferentiated Adipocytes to Insulin

Insulin promotes the differentiation of preadipocytes into adipocytes.The condition of obesity, which results in increases in fat cell number,occurs even in insulin-resistant states in which glucose transport isimpaired due to the antilipolytic effect of insulin. Inhibition oftriglyceride breakdown requires much lower insulin concentrations thanstimulation of glucose transport, resulting in maintenance or expansionof adipose stores (Kitamura et al., Mol. Cell. Biol., 1999, 19,6286-6296; Kitamura et al., Mol. Cell. Biol., 1998, 18, 3708-3717).

One of the hallmarks of cellular differentiation is the upregulation ofgene expression. During adipocyte differentiation, the gene expressionpatterns in adipocytes change considerably. Some genes known to beupregulated during adipocyte differentiation include hormone-sensitivelipase (HSL), adipocyte lipid binding protein (aP2), glucose transporter4 (Glut4), and peroxisome proliferator-activated receptor gamma(PPAR-γ). Insulin signaling is improved by compounds that bind andinactivate PPAR-γ, a key regulator of adipocyte differentiation(Olefsky, J. Clin. Invest., 2000, 106, 467-472). Insulin induces thetranslocation of GLUT4 to the adipocyte cell surface, where ittransports glucose into the cell, an activity necessary for triglyceridesynthesis. In all forms of obesity and diabetes, a major factorcontributing to the impaired insulin-stimulated glucose transport inadipocytes is the downregulation of GLUT4. Insulin also induces hormonesensitive lipase (HSL), which is the predominant lipase in adipocytesthat functions to promote fatty acid synthesis and lipogenesis(Fredrikson et al., J. Biol. Chem., 1981, 256, 6311-6320). Adipocytefatty acid binding protein (aP2) belongs to a multi-gene family of fattyacid and retinoid transport proteins. aP2 is postulated to serve as alipid shuttle, solubilizing hydrophobic fatty acids and delivering themto the appropriate metabolic system for utilization (Fu et al., J. LipidRes., 2000, 41, 2017-2023; Pelton et al., Biochem. Biophys. Res.Commun., 1999, 261, 456-458). Together, these genes play important rolesin the uptake of glucose and the metabolism and utilization of fats.

Leptin secretion and an increase in triglyceride content are alsowell-established markers of adipocyte differentiation. While it servesas a marker for differentiated adipocytes, leptin also regulates glucosehomeostasis through mechanisms (autocrine, paracrine, endocrine andneural) independent of the adipocyte's role in energy storage andrelease. As adipocytes differentiate, insulin increases triglycerideaccumulation by both promoting triglyceride synthesis and inhibitingtriglyceride breakdown (Spiegelman and Flier, Cell, 2001, 104, 531-543).As triglyceride accumulation correlates tightly with cell size and cellnumber, it is an excellent indicator of differentiated adipocytes.

The effect of antisense inhibition of C-reactive protein by on theexpression of markers of cellular differentiation was examined inpreadipocytes. Human white preadipocytes (Zen-Bio Inc., ResearchTriangle Park, N.C.) were grown in preadipocyte media (ZenBio Inc.,Research Triangle Park, N.C.). One day before transfection, 96-wellplates were seeded with approximately 3000 cells/well.

A randomized control oligonucleotide, ISIS 29848 (NNNNNNNNNNNNNNNNNNNN;where N is A, T, C or G; herein incorporated as SEQ ID NO: 617) was useda negative control, a compound that does not modulate adipocytedifferentiation. Tumor necrosis factor-alpha (TNF-α), which inhibitsadipocyte differentiation, was used as a positive control for theinhibition of adipocyte differentiation as evaluated by leptinsecretion. For all other parameters measured, ISIS 105990(AGCAAAAGATCAATCCGTTA, incorporated herein as SEQ ID NO: 622), aninhibitor of PPAR-γ, served as a positive control for the inhibition ofadipocyte differentiation. ISIS 29848 and ISIS 105990 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-O-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

Oligonucleotide was mixed with LIPOFECTIN™ reagent (Invitrogen LifeTechnologies, Carlsbad, Calif.) in OPTI-MEM™ medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) to acheive a final concentration of 250nM of oligonucleotide and 7.5 μg/mL LIPOFECTIN™ reagent. Before addingto cells, the oligonucleotide, LIPOFECTIN™ reagent and OPTI-MEM™ mediumwere mixed thoroughly and incubated for 0.5 hrs. Untreated control cellsreceived LIPOFECTIN™ reagent only. The medium was removed from theplates and the plates were tapped on sterile gauze. Each well was washedin 150 μl of phosphate-buffered saline. The wash buffer in each well wasreplaced with 100 μl of the oligonucleotide/OPTI-MEM™ medium/LIPOFECTIN™reagent cocktail. ISIS 133726 and ISIS 105990 were tested in triplicate,ISIS 29848 was tested in up to six replicate wells. The plates wereincubated for 4 hours at 37° C., after which the medium was removed andthe plate was tapped on sterile gauze. 100 μl of full growth medium wasadded to each well. After the cells have reached confluence(approximately three days), they were exposed for three days todifferentiation media (Zen-Bio, Inc.) containing a PPAR-γ agonist, IBMX,dexamethasone, and insulin. Cells were then fed adipocyte media(Zen-Bio, Inc.), which was replaced at 2 to 3 day intervals.

Leptin secretion into the media in which adipocytes are cultured wasmeasured by protein ELISA. On day nine post-transfection, 96-well plateswere coated with a monoclonal antibody to human leptin (R&D Systems,Minneapolis, Minn.) and left at 4° C. overnight. The plates were blockedwith bovine serum albumin (BSA), and a dilution of the treated adipocytemedia was incubated in the plate at room temperature for 2 hours. Afterwashing to remove unbound components, a second monoclonal antibody tohuman leptin (conjugated with biotin) was added. The plate was thenincubated with strepavidin-conjugated horseradish peroxidase (HRP) andenzyme levels were determined by incubation with3,3′,5,5′-tetramethlybenzidine, which turns blue when cleaved by HRP.The OD₄₅₀ was read for each well, where the dye absorbance isproportional to the leptin concentration in the cell lysate. Results,shown in Table 29, are expressed as a percent control relative tountreated control samples. With respect to leptin secretion, valuesabove or below 100% are considered to indicate that the compound has theability to stimulate or inhibit leptin secretion, respectively.

The triglyceride accumulation assay measures the synthesis oftriglyceride by adipocytes. Triglyceride accumulation was measured usingthe Infinity™ Triglyceride reagent kit (Sigma-Aldrich, St. Louis, Mo.).On day nine post-transfection, cells were washed and lysed at roomtemperature, and the triglyceride assay reagent was added. Triglycerideaccumulation was measured based on the amount of glycerol liberated fromtriglycerides by the enzyme lipoprotein lipase. Liberated glycerol isphosphorylated by glycerol kinase, and hydrogen peroxide is generatedduring the oxidation of glycerol-1-phosphate to dihydroxyacetonephosphate by glycerol phosphate oxidase. Horseradish peroxidase (HRP)uses H₂O₂ to oxidize 4-aminoantipyrine and 3,5 dichloro-2-hydroxybenzenesulfonate to produce a red-colored dye. Dye absorbance, which isproportional to the concentration of glycerol, was measured at 515 nmusing an UV spectrophotometer. Glycerol concentration was calculatedfrom a standard curve for each assay, and data were normalized to totalcellular protein as determined by a Bradford assay (Bio-RadLaboratories, Hercules, Calif.). Results, shown in Table 29, areexpressed as a percent control relative to untreated control samples.From these data, values for triglyceride (TRIG) accumulation above orbelow 100% are considered to indicate that the compound has the abilityto stimulate or inhibit triglyceride accumulation, respectively.

Expression of the four hallmark genes, HSL, aP2, Glut4, and PPARγ, wasalso measured in adipocytes transfected with compounds of the invention.Cells were lysed on day nine post-transfection, in aguanadinium-containing buffer and total RNA is harvested. The amount oftotal RNA in each sample was determined using a Ribogreen Assay(Invitrogen Life Technologies, Carlsbad, Calif.). Real-time PCR wasperformed on the total RNA using primer/probe sets for the adipocytedifferentiation hallmark genes Glut4, HSL, aP2, and PPAR-γ. mRNA levels,shown in Table 29, are expressed as percent control relative to theuntreated control values. With respect to the four adipocytedifferentiation hallmark genes, values above or below 100% areconsidered to indicate that the compound has the ability to stimulateadipocyte differentiation, or inhibit it, respectively.

TABLE 29 Effects of antisense inhibition of Tudor-SN on adipocytedifferentiation Treatment Leptin TRIG aP2 Glut4 HSL PPARγ ISIS 133726 8567 93 63 99 77 ISIS 29848 94 76 87 70 87 72 ISIS 105990 N.D. 38 55 53 5538 TNF-α 27 N.D. N.D. N.D. N.D. N.D.

ISIS 133726 reduced the expression levels leptin, triglycerides andGLUT4, suggesting that this antisense oligonucleotide is a candidateagent for applications where inhibition of adipocytes differentiation isdesirable, for example, obesity, hyperlipidemia, atherosclerosis,atherogenesis, diabetes, hypertension, or other metabolic diseases, aswell as having potential applications in the maintenance of thepluripotent phenotype of stem or precursor cells.

Example 43 Inflammation Assays Using Oligomeric Compounds Targeted toC-Reactive Protein

Inflammation assays are designed to identify genes that regulate theactivation and effector phases of the adaptive immune response. Duringthe activation phase, T lymphocytes (also known as T-cells) receivingsignals from the appropriate antigens undergo clonal expansion, secretecytokines, and upregulate their receptors for soluble growth factors,cytokines and co-stimulatory molecules (Cantrell, Annu. Rev. Immunol.,1996, 14, 259-274). These changes drive T-cell differentiation andeffector function. In the effector phase, response to cytokines bynon-immune effector cells controls the production of inflammatorymediators that can do extensive damage to host tissues. The cells of theadaptive immune systems, their products, as well as their interactionswith various enzyme cascades involved in inflammation (e.g., thecomplement, clotting, fibrinolytic and kinin cascades) representpotential points for intervention in inflammatory disease. Theinflammation assay presented here measures hallmarks of the activationphase of the immune response.

Dendritic cells treated with antisense compounds are used to identifyregulators of dendritic cell-mediated T-cell costimulation. The level ofinterleukin-2 (IL-2) production by T-cells, a critical consequence ofT-cell activation (DeSilva et al., J. Immunol., 1991, 147, 3261-3267;Salomon and Bluestone, Annu. Rev. Immunol., 2001, 19, 225-252), is usedas an endpoint for T-cell activation. T lymphocytes are importantimmunoregulatory cells that mediate pathological inflammatory responses.Optimal activation of T lymphocytes requires both primary antigenrecognition events as well as secondary or costimulatory signals fromantigen presenting cells (APC). Dendritic cells are the most efficientAPCs known and are principally responsible for antigen presentation toT-cells, expression of high levels of costimulatory molecules duringinfection and disease, and the induction and maintenance ofimmunological memory (Banchereau and Steinman, Nature, 1998, 392,245-252). While a number of costimulatory ligand-receptor pairs havebeen shown to influence T-cell activation, a principal signal isdelivered by engagement of CD28 on T-cells by CD80 (B7-1) and CD86(B7-2) on APCs (Boussiotis et al., Curr. Opin. Immunol., 1994, 6,797-807; Lenschow et al., Annu. Rev. Immunol., 1996, 14, 233-258).Inhibition of T-cell co-stimulation by APCs holds promise for novel andmore specific strategies of immune suppression. In addition, blockingcostimulatory signals may lead to the development of long-termimmunological anergy (unresponsiveness or tolerance) that would offerutility for promoting transplantation or dampening autoimmunity. T-cellanergy is the direct consequence of failure of T-cells to produce thegrowth factor IL-2 (DeSilva et al., J. Immunol., 1991, 147, 3261-3267;Salomon and Bluestone, Annu. Rev. Immunol., 2001, 19, 225-252).

Dendritic Cell Cytokine Production as a Measure of the Activation Phaseof the Immune Response

In a further embodiment of the present invention, the effect of ISIS133726 (SEQ ID NO: 36) was examined on the dendritic cell-mediatedcostimulation of T-cells. Dendritic cells (DCs, Clonetics Corp., SanDiego, Calif.) were plated at approximately 6500 cells/well on anti-CD3(UCHT1, Pharmingen-BD, San Diego, Calif.) coated 96-well plates in 500U/mL granulocyte macrophase-colony stimulation factor (GM-CSF) andinterleukin-4 (IL-4). DCs were treated with antisense compounds 24 hoursafter plating.

A randomized control oligonucleotide, ISIS 29848 (NNNNNNNNNNNNNNNNNNNN;where N is A, T, C or G; herein incorporated as SEQ ID NO: 617) servedas a negative control, a compound that does not affect dendriticcell-mediated T-cell costimulation. ISIS 113131 (CGTGTGTCTGTGCTAGTCCC,incorporated herein as SEQ ID NO: 623), an inhibitor of CD86, served asa positive control for the inhibition of dendritic cell-mediated T-cellcostimulation. ISIS 29848 and ISIS 113131 are chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings”. The wings arecomposed of 2′-O-methoxyethyl (2′-MOE) nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) throughout theoligonucleotide. All cytidine residues are 5-methylcytidines.

Oligonucleotide was mixed with LIPOFECTIN™ reagent (Invitrogen LifeTechnologies, Carlsbad, Calif.) in OPTI-MEM™ medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) to acheive a final concentration of 200nM of oligonucleotide and 6 μg/mL LIPOFECTIN™ reagent. Before adding tocells, the oligonucleotide, LIPOFECTIN™ reagent and OPTI-MEM™ mediumwere mixed thoroughly and incubated for 0.5 hrs. The medium was removedfrom the cells and the plates were tapped on sterile gauze. Each wellwas washed in 150 μl of phosphate-buffered saline. The wash buffer ineach well was replaced with 100 μL of the oligonucleotide/OPTI-MEM®medium/LIPOFECTIN™ reagent cocktail. Untreated control cells receivedLIPOFECTIN™ reagent only. ISIS 133726 and the positive control weretested in triplicate, and the negative control oligonucleotide wastested in up to six replicates. The plates were incubated witholigonucleotide for 4 hours at 37° C., after which the medium wasremoved and the plate was tapped on sterile gauze. Fresh growth mediaplus cytokines was added and DC culture was continued for an additional48 hours. DCs are then co-cultured with Jurkat T-cells in RPMI medium(Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10%heat-inactivated fetal bovine serum (Sigma Chemical Company, St. Louis,Mo.). Culture supernatants are collected 24 hours later and assayed forIL-2 levels (IL-2 DUOSET™ kit, R&D Systems, Minneapolis, Minn.), whichare expressed as a percent relative to untreated control samples. Avalue greater than 100% indicates an induction of the inflammatoryresponse, whereas a value less than 100% demonstrates a reduction in theinflammatory response.

The culture supernatant of cells treated with ISIS 133726, ISIS 29848and ISIS 113131 contained IL-2 at 84%, 83% and 55% of the IL-2concentration found in culture supernatant from untreated control cells,respectively. These results indicate that ISIS 133726 did not inhibitT-cell co-stimulation.

Cytokine Signaling as a Measure of the Effector Phase of theInflammatory Response

The cytokine signaling assay is designed to identify genes that regulateinflammatory responses of non-immune effector cells (initiallyendothelial cells) to both IL-113 and TNF-α (Heyninck et al., J CellBiol, 1999, 145, 1471-1482; Zetoune et al., Cytokine, 2001, 15,282-298). Response to cytokine stimulation is monitored by tracking theexpression levels of four genes: A20, intracellular adhesion molecule 1(ICAM-1), interleukin-9 (IL-8) and macrophage-inflammatory protein 2(MIP2α). As described below, these genes regulate numerous parameters ofthe inflammatory response. Antisense oligonucleotides are used toidentify genes that alter the cellular response to these cytokines.

A20 is a zinc-finger protein that limits the transcription ofpro-inflammatory genes by blocking TRAF2-stimulated NK-κB signaling.Studies in mice show that TNF-α dramatically increases A20 expression inmice, and that A20 expression is crucial for their survival (Lee et al.,Science, 2000, 289, 2350-2354).

ICAM-1 is an adhesion molecule expressed at low levels on restingendothelial cells that is markedly up-regulated in response toinflammatory mediators like tumor necrosis factor-α (TNF-α),interleukin-1β (IL-1β) and interferon-γ (IFN-γ) (Springer, Nature, 1990,346, 425-434). ICAM-1 expression serves to attract circulatingleukocytes into the inflammatory site.

IL-8 is a member of the chemokine gene superfamily, members of whichpromote the pro-inflammatory phenotype of macrophages, vascular smoothmuscle cells and endothelial cells (Koch et al., Science, 1992, 258,1798-1801). IL-8 has been known as one of the major inducible chemokineswith the ability to attract neutrophils to the site of inflammation.More recently, IL-8 has been implicated as a major mediator of acuteneutrophil-mediated inflammation, and is therefore a potentialanti-inflammatory target (Mukaida et al., Cytokine Growth Factor Rev,1998, 9, 9-23).

MIP2α, another chemokine known to play a central role in leukocyteextravasation, has more recently been shown to be involved in acuteinflammation (Lukacs et al., Chem Immunol, 1999, 72, 102-120). MIP2α isexpressed in response to microbial infection, to injection oflipopolysaccharides (LPS), and to stimulation of cells withpro-inflammatory mediators such as IL-1β and TNF-α (Kopydlowski et al.,J Immunol, 1999, 163, 1537-1544). Endothelial cells are one of severalcell types that are sources of MIP2α (Rudner et al., J Immunol, 2000,164, 6576-6582).

The effect of ISIS 133726 targeted to C-reactive protein was examined inhuman umbilical vascular endothelial cells (HUVECs) (ATCC, Manassus,Va.). HUVECs are cultured according to the supplier's recommendations.HUVECs are plated in a 96 well plate at a seeding density ofapproximately 3000 cells per well and are treated with antisensecompounds 24 hours later.

A randomized control oligonucleotide, ISIS 29848 (NNNNNNNNNNNNNNNNNNNN;where N is A, T, C or G; herein incorporated as SEQ ID NO: 617), wasused as a negative control, a compound that does not affect cytokinesignaling. ISIS 29848 is chimeric oligonucleotide (“gapmer”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-O-meLhoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines.

Oligonucleotide was mixed with LIPOFECTIN™ reagent (Invitrogen LifeTechnologies, Carlsbad, Calif.) in OPTI-MEM™ medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) to achieve a final concentration of 75nM of oligonucleotide and 2.25 μg/mL LIPOFECTIN™ reagent. Before addingto cells, the oligonucleotide, LIPOFECTIN™ reagent and OPTI-MEM™ mediumwere mixed thoroughly and incubated for 0.5 hrs. The medium was removedfrom the cells and the plates were tapped on sterile gauze. Each wellwas washed in 150 μl of phosphate-buffered saline. The wash buffer ineach well was replaced with 100 μL of the oligonucleotide/OPTI-MEM™medium/LIPOFECTIN™ reagent cocktail. Untreated control cells receivedLIPOFECTIN™ reagent only. ISIS 133726 was tested in triplicate, and ISIS29848 was tested in up to six replicate wells. The plates were incubatedwith oligonucleotide for 4 hours at 37° C., after which the medium wasremoved and the plate was tapped on sterile gauze. Fresh growth mediaplus cytokines was added and DC culture was continued for an additional46 hours, after which HUVECS were stimulated with 0.1 ng/mL of IL-1β or1 ng/mL TNF-α for 2 hours. Total RNA is harvested 48 hourspost-transfection, and real time PCR is performed using primer/probesets to detect A20, ICAM-1, IL-8 and MIP2α mRNA expression. Expressionlevels of each gene, shown in Table 30, are normalized to total RNA andvalues are expressed as a percent relative to untreated control samples.A value greater than 100% indicates an induction of the inflammatoryresponse, whereas a value less than 100% demonstrates a reduction in theinflammatory response.

TABLE 30 Effects of antisense inhibition of C-reactive protein on theinflammatory response +IL-1β +TNF α Treatment A20 ICAM-1 IL-8 MIP2α IL-8MIP2α ISIS 133726 95 64 77 58 130 77 ISIS 29848 101 89 96 86 84 71

ISIS 133726 inhibited the expression of ICAM-1, IL-8 and MIP2α inresponse to IL-1β stimulation, and therefore is a candidate agent forthe treatment of conditions in which inhibition or reduction of theinflammatory response is desirable, for example, in conditions such asrheumatoid arthritis, asthma and inflammatory bowel diseases.Conversely, ISIS 133726 stimulated the response of IL-8 in the presenceof TNF-α, suggesting that in this stimulatory pathway, inhibition ofC-reactive protein can stimulate an immune response, and is a candidateagent for the treatment of conditions in which stimulation of the immuneresponse is desirable, for example, in conditions characterized byimmunodeficiency.

Example 44 Antisense Oligonucleotides Targeted to Mouse C-ReactiveProtein In Vivo Lean Mouse Study

In a further embodiment, antisense oligonucleotides targeted to mouseC-reactive protein were tested for their effects on target expression,serum lipids, serum glucose and indicators of toxicity. Male C57Bl/6mice (Charles River Laboratories, Wilmington, Mass.) were fed a standardrodent diet. Mice were given intraperitoneal injections of 50 mg/kg ofeach of ISIS 147868 (SEQ ID NO: 580) and ISIS 147880 (SEQ ID NO: 592).Each oligonucleotide-treated group consisted of 5 mice. A total of 5saline-injected animals served as controls. Injections were administeredtwice weekly for a period of 2 weeks. At the end of the treatmentperiod, mice were sacrificed. No significant changes were observed inbody weights, which were recorded weekly, nor in liver and spleenweights recorded at necropsy.

C-reactive protein mRNA expression in liver was measured by real-timePCR, as described by other examples herein. ISIS 147868 and ISIS 147880,at a 50 mg/kg dose, resulted in 48% and 5% reductions in mouseC-reactive protein mRNA, respectively.

Serum was collected for routine clinical analysis of ALT, AST,cholesterol (CHOL), glucose (GLUC), HDL-cholesterol (HDL),LDL-cholesterol (LDL) and triglycerides (TRIG). These parameters weremeasured by routine procedures using an Olympus Clinical Analyzer(Olympus America Inc., Melville, N.Y.). The data are presented in Table31.

TABLE 31 Serum chemistry analysis of mice treated with antisenseoligonucleotides targeted to mouse C-reactive protein Serum parametersDose HDL LDL TG mg/ ALT AST CHOL mg/ mg/ mg/ GLUC Treatment kg IU/L IU/Lmg/dL dL dL dL mg/dL SALINE 27 62 80 61 11 102 243 147868 50 25 56 82 6112 113 214 147880 50 43 72 96 73 13 125 228

These data reveal that treatment with ISIS 147868 or ISIS 147880 did notresult in changes in the serum parameters measured. Together, theseresults illustrate that ISIS 147868 reduced C-reactive protein mRNAexpression in vivo without causing toxicity. ISIS 147880 did not causetoxicity in mice.

The invention claimed is:
 1. A modified compound 12 to 30 nucleobases inlength targeted to a nucleic acid molecule encoding C-reactive protein,wherein said compound is at least 90% complementary to said nucleic acidmolecule encoding C-reactive protein, wherein said compound comprises atleast an 8 nucleobase portion of a sequence selected from any of SEQ IDNOS:77, 78, 79, 82, 83, 86, 87, 88, 93, 94, 95, 96, 103, 104, 105, 106,107, 108, 109, 112, 113, 114, 115, 116, 117, 118, 119, 122, 123, 124,125, 126, 127, 128, 129, 133, 135, 136, 137, 138, 143, 144, 147, 148,158, 159, 160, 161, 162, 163, 167, 168, 169, 173, 177, 178, 179, 180,181, 189, 192, 193, 194, 195, 196, 199, 204, 206, 207, 208, 211, 213,214 and 215, wherein said modified compound comprises a modificationselected from the group consisting of a modified sugar and a modifiednucleobase, and wherein said compound inhibits the expression ofC-reactive protein mRNA.
 2. The compound of claim 1, wherein saidcompound is 15 to 30 nucleobases in length.
 3. The compound of claim 1,wherein said compound is an antisense oligonucleotide.
 4. The compoundof claim 1, wherein said compound is a chimeric oligonucleotide.
 5. Thecompound of claim 1 having at least 95%, at least 99% or 100%complementarity with said nucleic acid molecule encoding C-reactiveprotein.
 6. The compound of claim 1, having at least one modifiedinternucleoside linkage.
 7. The compound of claim 1, having at leastone: a) 2′-O-methoxyethyl sugar moiety; b) phosphorothioateinternucleoside linkage; or c) 5-methylcytosine.
 8. A method ofinhibiting the expression of C-reactive protein in a cell or tissuecomprising contacting said cell or tissue with the compound of claim 1so that expression of C-reactive protein is inhibited.
 9. A method oftreating an animal having a disease or condition associated withC-reactive protein comprising administering to said animal atherapeutically or prophylactically effective amount of the compound ofclaim 1 so that expression of C-reactive protein is inhibited.
 10. Themethod of claim 9, wherein the disease or condition is a cardiovasculardisorder.
 11. A method of inhibiting adipocyte differentiation inmammalian tissue comprising contacting said tissue with a compound ofclaim 1, wherein said compound inhibits the expression of C-reactiveprotein mRNA.
 12. The method according to claim 11, wherein saidmammalian tissue is tissue from a mammal having a metabolic disease. 13.The method according to claim 12, wherein said metabolic disease isselected from the group consisting of obesity, hyperlipidemia,atherosclerosis, atherogenesis, diabetes and hypertension.
 14. A methodfor maintaining the pluripotent phenotype of stem or precursor cellscomprising contacting said cells with a compound of claim 1, whereinsaid compound inhibits the expression of C-reactive protein mRNA.
 15. Amethod of inhibiting apoptosis in mammalian tissue comprising contactingsaid tissue with a compound of claim 1, wherein said compound inhibitsthe expression of C-reactive protein mRNA.
 16. The method according toclaim 15, wherein said mammalian tissue is tissue from a mammal having aneurodegenerative disorder.
 17. A method of inhibiting angiogenesis inmammalian tissue comprising contacting said tissue with a compound ofclaim 1, wherein said compound inhibits the expression of C-reactiveprotein mRNA.
 18. The method according to claim 17, wherein saidmammalian tissue is tissue from a mammal having a condition selectedfrom the group consisting of cancer, diabetic retinopathy,cardiovascular disease, rheumatoid arthritis and psoriasis.
 19. A methodof inhibiting or reducing inflammatory response in mammalian tissuecomprising contacting said tissue with a compound of claim 1, whereinsaid compound inhibits the expression of C-reactive protein mRNA. 20.The method according to claim 19, wherein said mammalian tissue is froma mammal having a disease selected from the group consisting ofrheumatoid arthritis, asthma and inflammatory bowel diseases.
 21. Amethod of treating a mammalian subject with an immunodeficiencycomprising administering to said mammal a compound of claim 1, whereinsaid compound inhibits the expression of C-reactive protein mRNA. 22.The compound of claim 1, wherein said compound is a chimericoligonucleotide 20 nucleotides in length, composed of a central gapregion consisting of ten 2′-deoxynucleotides, which is flanked on bothsides by five-nucleotide wings composed of 2′-O-methoxyethylnucleotides, wherein the internucleoside linkages are phosphorothioatethroughout the oligonucleotide, and all cytosine residues are5-methylcytosines.
 23. The method according to claim 9, wherein thedisease or condition is a cancerous disease or condition, an infectiousdisease or condition, a metabolic disease or condition, or aninflammatory disease or condition.
 24. The compound according to any ofclaim 1, 2, 3 or 4, for use in treating a neurological condition, ametabolic condition, a cardiovascular condition, a women's healthcondition, an inflammatory disease, an infectious disease, a pulmonarycondition, a musculoskeletal condition, or cancer, wherein: (a) theneurological condition is obstructive sleep apnea, Alzheimer's disease,ALS, Parkinson's disease, ataxia, or macular degeneration; (b) themetabolic condition is obesity, metabolic syndrome, or diabetes; (c) thecardiovascular condition is a risk of sudden cardiac death, coronaryartery disease (CAD), unstable angina, stroke, elective stent placement,angioplasty, atherosclerosis, percutaneous transluminal angioplasty(PTCA), peripheral vascular disease, myocardial infarction (MI), cardiactransplantation, hypertension, mitral valve commissurotomy, thrombosis,deep vein thrombus, end-stage renal disease (ESRD), renal dialysis,complement activation, congestive heart failure, systemic vasculitis,cardiopulmonary bypass, hyperlipidemia, acute coronary syndrome orcoronary artery stenting; (d) the women's health condition ispremenstrual syndrome (PMS) or dysmenorhhoea; (e) the inflammatorydisease is gingivitis, inflammatory bowel disease, ulcerative colitis,rheumatoid arthritis, osteoarthritis, or axial spondyloarthritis; (f)the infectious disease is HW-associated rheumatic disorder or bacterialinfection; (g) the pulmonary condition is asthma or chronic obstructivepulmonary disease; (h) the musculoskeletal condition is lower back pain,intense physical exercise, endurance training, or age-related disorders;or (i) the cancer is pulmonary cancer or colon cancer.
 25. Apharmaceutical composition comprising the compound of claim 1, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable diluent or carrier.